Peptides of Syndecan-1 For Inhibiting Angiogenesis

ABSTRACT

The present invention provides a peptide derived from the extracellular domain of syndecan-1 that inhibits angiogenesis.

The present application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 60/812,187, filed Jun. 9, 2006, the entire contentsof which are hereby incorporated by reference.

The government own rights to the present invention pursuant to fundingfrom the National Institute of Health grant numbers R01-HD21881 andCA109010.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of proteinchemistry and developmental biology. More particularly, it concernspeptide segments from the extracellular domain of syndecan-1 (Sdc-1)that can inhibit angiogenesis and can thus be used to treat angiogenesisin pathologic conditions.

II. Description of Related Art

A. Function of α_(v)β₃ and α_(v)β₅ Integrins in Angiogenesis

Several different growth factors, among them fibroblast growth factor(FGF) and vascular endothelial cell growth factor (VEGF), are oftenreleased by tumors to cause endothelial cells to undergo angiogenesis.Blood vessels in the vicinity of the tumor respond to VEGF by becomingleaky (thus the alternate name “vascular permeability factor”) allowingfibronectin, vitronectin and fibrinogen in the blood to infiltrate thesurrounding matrix. These matrix ligands are critical adhesion andactivation ligands for the αvβ3 and αvβ5 integrins, which have roles inthe chemotactic migration of the endothelial cells and in the survivalof the cells during vessel pruning. A second response to the growthfactors, particularly FGF, is the activation of a neovessel developmentprogram that relies on Hox master genes (Boudreau et al., 1997; Myers etal., 2000; Myers et al., 2002). HoxD3 is initially expressed andcontrols a family of genes that are necessary for the initial migrationprocess, including upregulation of the αvβ3 integrin, MMPs and uPAR(Boudreau et al., 1997). Expression of HoxD3 is followed by HoxB3, whichregulates the morphogenesis leading to the formation of small vessels(Myers et al., 2000), and finally by the HoxD10 gene, which restores themature phenotype of the cells (Myers et al., 2002); it is HoxD10 that isexpressed in resting, stable blood vessels in vivo.

The αvβ3 and αvβ5 integrins are important not only during endothelialcell migration, but are important players in the survival of theendothelial cells. Although endothelial cells in mature vessels are notreadily susceptible to apoptosis, angiogenic cells that are induced bygrowth factors are highly susceptible and rely upon the continuedpresence of these factors for survival. This is shown experimentally byinducing angiogenesis with VEGF and causing apoptosis by its withdrawal,and is demonstrated in vivo when the developing ovarian follicle inducesangiogenesis by release of VEGF and the newly formed bloods vesselsregress when the source of VEGF is lost upon ovulation. Endothelialcells responding to VEGF rely on signaling from the αvβ5 integrin inorder to prevent this apoptotic process (Brooks et al., 1994;Friedlander et al., 1995). Similarly, endothelial cells responding tofibroblast growth factor (FGF) are susceptible to apoptosis unless thereis coordinate signaling from the αvβ3 integrin. Thus, inhibitors thatdisrupt the activation of these two integrins are potential drugs forblocking angiogenesis not only because they can prevent the positivesignaling from the integrins that aid in endothelial cell migration andformation of new vessels, but also because they have the potential toelicit “negative” signals that trigger apoptosis and death of theendothelial cell. Furthermore, this mechanism is not confined toendothelial cells and would apply as well to tumor cells or other cellsthat rely on either of these integrins to initiate disease processes.

B. Syndecans

The syndecan family of cell surface heparan sulfate (HS) proteoglycansis comprised of four vertebrate members. These receptors are expressedon virtually all cell types, although their expression may be altered indisease states such as cancer (Beauvais and Rapraeger, 2004). Thesyndecan core proteins share a high degree of conservation in theirshort cytoplasmic and transmembrane (TM) domains; in contrast, theirectodomains (EDs) are divergent with the exception of attachment sitesfor HS glycosaminoglycans. Via their HS chains, syndecans regulate thesignaling of growth factors, chemokines, and morphogens and engagecomponents of the ECM including VN, FN, LN, tenascin, thrombospondin,and the fibrillar COLs (Bernfield et al., 1999).

In addition to the activities of their HS chains, the syndecan coreproteins have roles in cell adhesion signaling (Rapraeger, 2000; Tumovaet al., 2000). Conserved and variable regions of the syndecancytoplasmic domains appear critical for binding interactions that leadto adhesion-mediated signaling and reorganization of the actincytoskeleton (Couchman et al., 2001). Important roles for the TM domainhave also been demonstrated for Sdc-1 and syndecan-4 (Sdc-4) (Tkachenkoand Simons, 2002; McQuade and Rapraeger, 2003). Perhaps the leastexpected active protein domain is the syndecan ED, which bears the HSchains. Nonetheless, several emerging studies suggest that the syndecanED may have important regulatory roles in cell adhesion signaling. Cellspreading and morphogenetic activities in COS-7 and Schwann cells tracein part to the S1ED (Carey et al., 1994; Adams et al., 2001). Raji cellsrequire the Sdc-1 TM domain for initial spreading, but depend on a S1EDactivity for cell polarization (McQuade and Rapraeger, 2003). Moreover,inhibition of ARH-77 myeloma and hepatocellular carcinoma cell invasioninto a COL I matrix by Sdc-1 also traces to a region of itsextracellular core protein domain (Liu et al., 1998; Ohtake et al.,1999).

The activities of other syndecans also trace to their EDs.Overexpression of Syndecan-2 (Sdc-2) in COS-1 and Swiss 3T3 cellsinduces filipodial extension and deletion mutants of Sdc-2 map activityto the S2ED (Granes et al., 1999). Upregulation of Sdc-2 expression incolon carcinoma cells leads to altered cell morphology and colonyformation in soft agar; treatment with recombinant S2ED disrupts thesebehaviors (Park et al., 2002; Kim et al., 2003). Finally, activatedB-lymphocytes, when seeded on S4ED antibodies, exhibit morphologicalchanges and filipodial extensions. Intriguingly, only the S4ED isrequired for this response, indicating that it may interact with a TMpartner to transmit a dendritic signal (Yamashita et al., 1999).

C. α_(v)β₃ Integrins are Regulated by Syndecan-1

The inventors' previous work in the MDA-MB-231 cells suggested that cellspreading induced upon anchorage of the cells to a Sdc-1 antibody relieson functional coupling of the syndecan to activated α_(v)β₃ integrins(Beauvais and Rapraeger, 2003). This spreading response is rapid (˜15-30min) and occurs even in the absence of an integrin ligand (i.e.,spreading is not blocked by cycloheximide or EGTA treatment), so long asthe cells are adherent via Sdc-1. Intriguingly, the α_(v)β₃-dependentspreading mechanism is blocked by the addition of soluble, recombinantSdc-1ED, suggesting that anchorage of Sdc-1 to a ligand provides aplatform for α_(v)β₃ integrin activation and adhesion signaling viabinding interaction of the syndecan ED. These findings raised afundamental question about the role of Sdc-1 in ECM signaling, inparticular whether or not Sdc-1 is required for α_(v)β₃ activation andsignaling in response to a native matrix ligand. The inventors went on,in subsequent studies to show that Sdc-1 is required for signalingthough both α_(v)β₃ and α_(v)β₅, and that inhibition of this function bycompetitive binding (Sdc-1ED) or siRNA inhibition blocks cellattachment, cell spreading, cell migration and angiogenesis(unpublished).

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided anisolated and purified peptide or polypeptide segment consisting ofbetween 5 and 100 amino acid residues and comprising SEQ ID NO:21 or SEQID NO:13. In certain embodiments, the peptide does not have an aminoacid sequence that consists of SEQ ID NO:28, that is, the peptidediffers from SEQ ID NO:28.

The peptide or polypeptide may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acidresidues in length, or any range derivable therein. In some embodiments,the peptide or polypeptide is between 10 and 80, 20 and 50, or 30 and 40amino acid residues in length.

In some embodiments, the peptide may consist of SEQ ID NO:1 or SEQ IDNO:10. In other embodiments, the peptide may consist of or comprise SEQID NO:1, SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:28.

In further embodiments, the peptide or polypeptide comprises at least orat most 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 47, 48, or 49contiguous amino acids from SEQ ID NO:10, or any range derivabletherein. In additional embodiments, the peptide or polypeptide comprisesat least or at most 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 47, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 contiguous amino acidsfrom SEQ ID NOS:14, 16, 18, or 20, or any range derivable therein. It isalso contemplated that in certain embodiments, the peptide orpolypeptide is at least 90%, 85%, 90%, 95%, or 100% identical to anyspecified length of a peptide based on SEQ ID NOS;14, 16, 18, or 20. Forinstance, peptides or polypeptides of the invention may be 30-40 aminoacids in length with at least 90% identity to a sequence of that lengthfrom SEQ ID NO:20.

It is specifically contemplated that any embodiment relating to apeptide or polypeptide of the invention may be implemented in any otherembodiment of the invention, including in methods of the invention.

In another embodiment, there is provided a nucleic acid encoding apeptide or polypeptide segment consisting of between 5 and 100 aminoacid residues and comprising SEQ ID NO:10. The nucleic acid may encode apeptide or polypeptide of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues inlength, or any range derivable therein. The nucleic acid encodes apeptide consisting of SEQ ID NO:10. In other embodiments, the nucleicacid encodes a peptide consisting of or comprising SEQ ID NO:1, SEQ IDNO:13, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:28.

In yet another embodiment, there is provided a recombinant cellcomprising a nucleic acid encoding a peptide or polypeptide segmentconsisting of between 5 and 100 amino acid residues and comprising SEQID NO:21 or SEQ ID NO:13, or any amino acid segment discussed above. Thecell may be a bacterial cell. The cell may comprise a nucleic acidfurther encoding a peptide tag fused to the nucleic acid encoding saidpeptide segment.

In still yet another embodiment, there is provided a pharmaceuticalcomposition comprising an isolated and purified peptide or polypeptidesegment consisting of between 5 and 100 amino acid residues andcomprising SEQ ID NO:21 or SEQ ID NO:13 (or any amino acid segmentdiscussed above) dispersed in a pharmaceutically acceptable buffer ordiluent. In certain embodiments, the peptide does not have an amino acidsequence that consists of SEQ ID NO:28.

In a further embodiment, there is provided a method of inhibitinginteraction of α_(v)β₃ or α_(v)β₅ integrin with syndecan-1 comprisingcontacting a α_(v)β₃ or α_(v)β₅ integrin molecule with a peptide orpolypeptide segment consisting of between 5 and 100 amino acid residuesor less residues and comprising SEQ ID NO:21 or SEQ ID NO:13. Thepeptide or polypeptide may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 amino acid residues inlength, or any range derivable therein. In certain embodiments, thepeptide does not have an amino acid sequence that consists of SEQ IDNO:28. The peptide may consist of SEQ ID NO:10. In other embodiments,the peptide may consist of or comprise SEQ ID NO:1, SEQ ID NO:13, SEQ IDNO:21, SEQ ID NO:23, or SEQ ID NO:28. The α_(v)β₃ or α_(v)β₅ integrinmay be located on the surface of a cell, such as a cancer cell (e.g., acarcinoma, a myeloma, a melanoma or a glioma), including a metastaticcancer cell. The method may further comprise contacting said cancer cellwith a second cancer inhibitory agent.

In yet a further embodiment, there is provided a method of inhibitingα_(v)β₃ or α_(v)β₅ integrin activation by syndecan-1 comprisingcontacting a cell expressing an α_(v)β₃ or α_(v)β₅ integrin moleculewith a peptide or polypeptide segment consisting of between 5 and 100amino acid residues and comprising SEQ ID NO:21 or SEQ ID NO:13. Incertain embodiments, the peptide does not have an amino acid sequencethat consists of SEQ ID NO:28. The peptide or polypeptide may be 10, 15,20, 25, 30, 35, 40, 45, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or100 amino acid residues in length. The peptide may consist of SEQ IDNO:10. In other embodiments, the peptide may consist of or comprise SEQID NO:1, SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:28. Theinhibiting may result in inhibition of cell adhesion, migration, cellmetastasis, cell survival and/or cell proliferation.

In still yet a further embodiment, there is provided a method oftreating a subject with a cancer, cells of which express α_(v)β₃ orα_(v)β₅ integrin, comprising contacting said cells with a peptide orpolypeptide segment consisting of between 5 and 100 amino acid residuesor less residues and comprising SEQ ID NO:21 or SEQ ID NO:13. In certainembodiments, the peptide does not have an amino acid sequence thatconsists of SEQ ID NO:28. The peptide or polypeptide may be 10, 15, 20,25, 30, 35, 40, 45, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100amino acid residues in length. The peptide may consist of SEQ ID NO:10.In other embodiments, the peptide may consist or comprise SEQ ID NO:1,SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:28. The subjectmay be a human. The cancer may be a carcinoma, a myeloma, a melanoma ora glioma. The peptide or polypeptide may be administered directly tosaid cancer cells, local to said cancer cells, regional to said cancercells, or systemically. The method may further comprise administering tosaid subject a second cancer therapy selected from chemotherapy,radiotherapy, immunotherapy, hormonal therapy, or gene therapy.

In an additional embodiment, there is provided a method of inhibitingangiogenesis comprising contacting an endothelial cell expressing anα_(v)β₃ or α_(v)β₅ integrin molecule with a peptide or polypeptidesegment consisting of between 5 and 100 amino acid residues andcomprising SEQ ID NO:21 or SEQ ID NO:13. In certain embodiments, thepeptide does not have an amino acid sequence that consists of SEQ IDNO:28. The peptide or polypeptide, may be 10, 15, 20, 25, 30, 35, 40,45, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acidresidues in length. The peptide may consist of SEQ ID NO:10. In otherembodiments, the peptide may consist of or comprise SEQ ID NO:1, SEQ IDNO:13, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:28.

In still an additional embodiment, there is provided a method oftreating a subject having a disease characterized by angiogenesiscomprising contacting endothelial cells which express α_(v)β₃ or α_(v)β₅integrin and are responsible for said angiogenesis, with a peptide orpolypeptide segment consisting of between 5 and 100 amino acid residuesor less residues and comprising at least or at most 10, 11, 12, 13, 14,15, 16, 171, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or32 contiguous amino acids from a synstatin sequence (or any rangederivable therein), such as those defined by SEQ ID NOS:15, 17, 19, and21. In certain embodiments, the segment comprises SEQ ID NO:21 or SEQ IDNO:13. In certain embodiments, the peptide does not have an amino acidsequence that consists of SEQ ID NO:28. The peptide or polypeptide maybe 10, 15, 20, 25, 30, 35, 40, 45, 49, 50, 55, 60, 65, 70, 75, 80, 85,90, 95 or 100 amino acid residues in length. The peptide may consist ofSEQ ID NO:10. In other embodiments, the peptide may consist of orcomprise SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:23, or SEQID NO:28. The disease may be an abnormality of the vasculature(atherosclerosis and hemangiomas), of the eye (diabetic retinopathy andretinopathy of prematurity), of the skin (pyogenic granulomas,psoriasis, warts, scar keloids, allergic edema, ulcers), of the uterusand ovary (dysfunctional uterine bleeding, follicular cysts,endometriosis, pre-eclampsia), of the adipose tissue (obesity), of thebones and joints (rheumatoid arthritis, osteophyte formation), andAIDS-related pathologies resulting from TAT protein of the humanimmunodeficiency virus (HIV) activating the avb3 integrin on endothelialcells. In certain embodiments, the disease is particularly not cancer.The contacting may comprise systemic administration of said peptide orpolypeptide or administration of said peptide or polypeptide local tosaid endothelial cells. The peptide or polypeptide may be active at 0.3μM, at 0.2 μM or at 0.1 μM, or between 0.1 μM and 0.3 μM. The method mayfurther comprise contacting said endothelial cells with a secondanti-angiogenic agent.

It is further contemplated that peptides or polypeptides of theinvention may also include amino acid segments from other proteins.These peptides and polypeptides would have an amino acid sequence from anon-syndecan-1 protein. Consequently, peptides or polypeptides of theinvention may consist essentially of a syndecan-1 amino acid segment,such as those discussed herein, in which case they can have an aminoacid segment from another protein. In particular embodiments, the otherprotein can be some kind of marker, targeting sequence, or stabilizer.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.” The word “about” means plus or minus 5% ofthe stated number.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed.

FIGS. 1A-C—Co-expression of Sdc1, α_(v)β₃ integrin and α_(v)β₅ integrinby endothelial cells during tumor-induced angiogenesis. (FIG. 1A) Wnt-1induced mammary tumor. (FIG. 1B) β-catenin-induced mammary tumor. (FIG.1C) Normal mouse artery. Endothelial cells are identified by stainingwith a rat monoclonal for PECAM. The integrin subunits are identified bystaining with rabbit polyclonal antibodies to the β3 subunit (AB1932),β5 subunit (AB1926) and αV subunit (AB1930). Sdc1 is visualized by anaffinity purified rabbit polyclonal antibody directed against itsextracellular domain. Rat IgG and Rabbit IgG controls are shown. Thestaining in the artery is shown as an overlay of the two antibodiesused. Note that neither Sdc1 nor the two integrins are expressed in thePECAM-positive endothelial cell layer, whereas they are in the tumors.

FIG. 2—Human aortic endothelial cells rely on αvβ₃ and αvβ₅ integrinsfor spreading on VN. Human aortic endothelial cells (HAECs) weresuspended and replated on 10 μg/ml VN in the presence of integrininhibitory antibodies mAb 13 (specific for β1-containing integrins; thiswill inhibit the αvβ1 integrin), LM609 (specific for the αvβ₃ integrin)and P1F6 (specific for the αvβ₅ integrin), or combinations of thesemAbs. After 2 hr, the cells were fixed, stained with Alexa488-conjugatedphalloidin to aid in visualization, and observed by fluorescencemicroscopy to document integrin-dependent spreading. Inhibition ofspreading requires simultaneous inhibition of both αvβ₃ and αvβ₅integrins.

FIG. 3—αvβ₃ and αvβ₅ integrin-mediated spreading of human aorticendothelial cells on VN is disrupted by recombinant Sdc1 ectodomainfusion protein. HAECs were plated on VN in the presence of nocompetitor, 1-10 μM GST-mS1ED (recombinant mouse Sdc1 ectodomainexpressed as a fusion protein with GST) or GST alone.

FIG. 4—Inhibition of HAEC spreading on VN by silencing of Sdc-1. HAECswere transfected with a range of concentrations of siRNA specific forhuman Sdc-1 (hS1), which achieves 80-88% silencing at 100-200 nM, asshown by flow cytometry using human Sdc-1 mAb B-B4. The cells treatedwith 200 nM siRNA were also plated on VN for 2 hr and display a 60%reduction in cell spreading.

FIGS. 5A-D—Inhibition of MDA-MB-231 human mammary carcinoma cellattachment and spreading on VN by synstatin₈₂₋₁₃₀. MDA-MB-231 humanmammary carcinoma cells, which rely on the αvβ₃ integrin for attachmentand spreading on VN, were plated on 10 μg/ml VN for 2 hr in the absenceof peptide (FIG. 5A) or in the presence of 0.1 (FIG. 5B), 0.3 (FIG. 5C)or 1 μM (FIG. 5D) synstatin₈₂₋₁₃₀. The cells were fixed and stained asin FIG. 2.

FIGS. 6A-H—Inhibition of B82L fibroblast and HMEC-1 cell attachment andspreading on VN by synstatin₈₂₋₁₃₀. B82L fibroblasts, which rely on theαvβ₅ integrin for attachment to VN, and human dermal microvascularendothelial cells, which rely on both the αvβ₃ and αvβ₅ integrins forattachment to VN, were plated in the absence of peptide (FIGS. 6A, E) orin the presence of 0.1 (FIGS. 6B, F), 0.3 (FIGS. 6C, G) or 1 μM (FIGS.6D, H) synstatin₈₂₋₁₃₀ for 2 hr and processed as described in FIG. 2.

FIGS. 7A-D—Synstatin82-130 inhibits angiogenesis induced by FGF in vivo.A polyHEMA pellet containing 100 ng FGF was surgically inserted into thecenter of the mouse cornea. Test mice also received a 2 ml Alzet osmoticpump inserted subcutaneously on their backs, containing 100 μMsynstatin₈₂₋₁₃₀. The mice were allowed to recover and were maintainedfor seven days before sacrifice. Fluorescent dextran was injected intothe vascular system via retro-orbital injection just prior to sacrifice.(FIG. 7A) mouse with FGF alone; (FIG. 7B) fluorescence image of theboxed area shown in FIG. 7A; (FIG. 7C) mouse with FGF+systemicsynstatin₈₂₋₁₃₀; (FIG. 7D) fluorescence image of boxed area shown inFIG. 7C. Note that the cornea is devoid of angiogenic vessels in thepresence of systemic synstatin. Normal vessels are observed in the iris.

FIGS. 8A-D—SSTN disrupts association of syndecan-1 with the α_(v)β₃ andα_(v)β₅ integrins. (FIG. 8A) Diagram of mouse syndecan-4 (mS4), humansyndecan-1 (hS1) and mouse syndecan-1 (mS1) and mS1 deletion mutantsshowing which proteoglycans are able to activate the α_(v)β₃ and α_(v)β₅integrins. (FIG. 8B) Flow cytometry of human aortic endothelial cells(HAEC), human dermal microvascular endothelial cells (HMEC-1) or mouseaortic endothelial cells (MAEC) using human or mouse-specific antibodiesfor syndecan-1, α_(v)β₃, and α_(v)β₅. The β₅ subunit is detected byWestern blot in mouse cells and is compared to B82L fibroblasts known toexpress this integrin; (FIG. 8C) Human syndecan-1 is immunoprecipitatedfrom HMEC-1 cells in the presence or absence of 30 μM GST, 30 μMGST-mS1ED (mouse syndecan-1 ectodomain), or 1 μM SSTN and detected onblots along with co-precipitating β₃ or β₅ integrin. (FIG. 8D) Humansyndecan-1 is immunoprecipitated from MDA-MB-231 human mammary carcinomacells expressing full-length mouse syndecan-1 (Fl-mS1), a mouse mutantbearing deletion of amino acids 67-121 (mS1^(Δ67-121)), or vector alone(NEO) and blotted for the co-precipitation of the α_(v)β₃ integrin bydetection of the β₃ subunit.

FIG. 9—Sequence of SSTN across species. The sequence of syndecan-1 inthe vicinity of the α_(v)β₃ and α_(v)β₅ regulatory site is shown formurine, hamster, rat and human. The numbering is for the murinesequence. The active site defined by deletion analysis is amino acids88-121 in the mouse sequence. Note that human syndecan-1 has oneadditional amino acid N-terminal to the SSTN sequence; thus itsnumbering would be greater by one amino acid. A putative conservedsequence (cons) is shown (SEQ ID NO:13) (72% homology).

murS1 shown is SEQ ID NO:14 and synstatin sequence is SEQ ID NO:15.

hamS1 is SEQ ID NO:16 and synstatin sequence is SEQ ID NO:17.

ratS1 is SEQ ID NO:18 and synstatin sequence is SEQ ID NO:19.

humS1 is SEQ ID NO:20 and synstatin sequence is SEQ ID NO:21.

FIGS. 10A-C—Dependence of HMEC cell attachment and spreading on integrinactivation by Sdc1 and its inhibition by SSTN. (FIG. 10A) HMECs wereplated for 2 hr either on Sdc1-specific antibody B-B4 to engage Sdc1 oron VN (10 μg/ml coating concentration) to engage the α_(v)β₃ and α_(v)β₅integrins. Blocking antibodies LM609 (10 μg/ml, specific for α_(v)β₃)and P1F6 (10 μg/ml, specific for α_(v)β₅), SSTN (0.5 μM) or recombinantmouse Sdc1 ectodomain (mS1ED, 5 μM) were added. The ligand mimeticantibody WOW1 was used to detect activated α_(v)β₃ integrin on cellsplated on B-B4. (FIG. 10B) HMECs were treated with siRNA oligo specificfor human Sdc1 for 48 hr prior to plating on VN. Sdc1 expression wasquantified by flow cytometry using human Sdc1-specific mAb B-B4 or acontrol mouse IgG. (FIG. 10C-D) The percentage of cell attaching orattaching and spreading was quantified for HMECs, HAEC and MAECs platedon VN for 24 hr in the presence of mS1ED or SSTN, or after siRNAsilencing of Sdc1 expression in the presence of mAb LM609 to blockα_(v)β₃ integrin, P1F6 to block α_(v)β₅ integrin, or both antibodies.

FIGS. 11A-C—Syndecan-1 and the α_(v)β₃ and α_(v)β₅ integrin areco-expressed during aortic angiogenesis and in tumors. (FIG. 11A) Aorticexplants were stained en face for expression of syndecan-1, α_(v)β₃ andα_(v)β₅ integrin (α_(v), β₃ and β₅) and PECAM. (FIG. 11B) Aorticsegments explanted to collagen gels in the presence of 30 ng/mlfibroblast growth factor-2 (FGF-2) were stained after 7 days forexpression of syndecan-1 and β₃ or β₅ integrin. (FIG. 11C) Frozensections of spontaneous mouse mammary tumors from the wnt-1 or β-cateninoverexpressing mice were stained for syndecan-1, PECAM, of integrinexpression (α_(v), β₃, β₅).

FIG. 12—SSTN blocks aortic ring angiogenesis. Aortic segments wereexplanted to collagen gels for 7 days in the presence of 50 ng/mlvascular endothelial cell growth factor (VEGF), or 30 ng/ml FGF-2 in thepresence or absence of mS1ED or SSTN. Total outgrowth in quantified asthe total length of all microvessels.

FIG. 13—Inhibition of corneal angiogenesis by SSTN. PolyHEMA pellets(0.25 μL) containing 67 ng FGF-2 and sucralfate were implanted into theavascular mouse cornea. Alzet pumps containing PBS, 2500 μM mS1ED or arange of SSTN concentrations (0-100 μM) were implanted subcutaneously onthe back of the animals and angiogenesis for 7 days. Fluorescent dextranwas injected suborbitally for 2 min prior to sacrifice for observationof the vascular system in dissected cornea (picture insets). Totalangiogenesis is quantified as total length of vessels growing from thelimbic vessels at the margin of the cornea.

FIG. 14—Activity of SSTN peptides. A diagram of SSTN₈₂₋₁₃₀ used in ourexperiments is shown. The gray letters designate amino acids that areidentical or highly conserved across species (mouse, human, rat,hamster). Shorter peptides have been tested using HMEC-1 endothelialcells for inhibition of α_(v)β₃ and α_(v)β₅ mediated cell attachment andspreading in comparison to SSTN₈₂₋₁₃₀.

SSTN₈₂₋₁₃₀ (49-mer) is SEQ ID NO:22. SSTN₈₈-122 (35-mer) is SEQ IDNO:23. SSTN₉₆₋₁₃₀ (35-mer) is SEQ ID NO:24. SSTN₈₈₋₁₁₇ (30-mer) is SEQID NO:25. SSTN₈₈₋₁₀₆ (19-mer) is SEQ ID NO:26. SSTN₉₀₋₁₀₃ (14-mer) isSEQ ID NO:27.

FIG. 15—SSTN-treated mice bearing CAG human myeloma tumors. 10⁵ humanCAG myeloma cells were injected subcutaneously into the backs ofimmunodeficient SCID mice. After 10 days, the mice were scanned toconfirm establishment of luciferase-expressing tumors in all ten mice.Alzet pumps delivering 0.25 μl/hr of either PBS (control) or 100 μM SSTNwere implanted and the tumors allowed to grow for an additional 28 days.Scanning of the tumors at this point (measured asphotons/second/cm2/steradian) shows an 11-fold reduction in tumor sizeby treatment with SSTN (not shown.) The five mice on the right receivedPBS and have visible, large tumors (circled). The mice on the leftreceived SSTN and have smaller tumors. The pump is visible on all miceas a purple spot on their right flank.

FIGS. 16A-B—Myeloma tumors from SSTN-treated mice. (FIG. 16A) Myelomatumors from either PBS or SSTN treated mice were dissected and weighedafter 28 days: control (238+/−92 g) and SSTN-treated (22+/−6 g) tumorsagain showing an 11-fold difference (P=0.02). (FIG. 16B) A PBS controland a SSTN-treated tumor were sectioned and stained with mouse-specificanti-CD34 to view in the ingrowth of host blood vessels to the humantumor. The control tumors had more numerous and much longer vessels(arrowheads) than the treated tumors. There was no positive stainingusing human-specific anti-CD34 (not shown), indicating that thevasculature in the tumors is due to host angiogenesis.

FIG. 17—Human SSTN peptides inhibit α_(v)β₃-mediated cell attachment andspreading on vitronectin. MDA-MB-231 human mammary carcinoma cells wereallowed to attach and spread on either vitronectin (VN) or fibronectin(FN) during a 2 hr assay in the presence of 0, 0.1, 0.3, 0.5, 1.0 or 30μM SSTN peptide. The 30 μM concentration was used only for FN todemonstrate its lack of activity on this substratum. The two peptidestested are derived either from amino acids 88-121 (hSSTN 88-121) or89-120 (hSSTN 89-120) of the human Sdc1 sequence. Adhesion to VN, butnot FN, is mediated by the α_(v)β₃ integrin on these cells and isdisrupted by the human SSTN peptides.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. The Present Invention

The inventors previously established a link between Sdc-1 and theα_(v)β₃ integrin (Beauvais and Rapraeger, 2003). Subsequently, they alsoshowed that addition of recombinant mouse Sdc-1 (mS1) ectodomain (ED) oranti-Scd-1ED polyclonal antibodies (pAbs) block integrin activation anddisrupt α_(v)β₃-dependent spreading and migration of cancer cells, andthat downregulation of human Sdc-1 (hS1) expression by small-interferingRNA (siRNA) disrupts cancer cell spreading and migration on VN, but noton FN. These data suggest that Sdc-1 and the α_(v)β₃ integrin arefunctionally coupled via the Scd-1ED and that coupling is required forα_(v)β₃ integrin activation and signaling.

In addition, the inventors demonstrated a syndecan link to α_(v)β₅integrin. The inventors showed that B82L fibroblast cells rely almostsolely on the α_(v)β₅ integrin for attachment and spreading on VN andthis integrin activity depends on Sdc-1. Cells ligated by Sdc-1 antibodydisplayed hyperactivation of the α_(v)β₅ integrin. In addition,expression studies showed that the expression of the extracellulardomain of Sdc-1 at the cell surface is necessary for integrinactivation. In keeping with this finding, competition with therecombinant ectodomain of Sdc-1 inactivated the integrin, as shown bythe failure of cells to attach and spread to the α_(v)β₅ integrin ligandVN.

The formation of new blood vessels, which occurs in development anddisease, relies on inducing proliferation and migration of endothelialcells, and in controlling the survival or apoptosis of the cells tosculpt the architecture of the new vessels (vascular pruning). The αvβ3and αvβ5 integrins have important roles in all three of these steps.Surprisingly, work with β3, β5, or double β3/β5 knockout mice shows thatangiogenesis in mice lacking one or both of these integrins is notblocked, and may be increased (Reynolds et al., 2002). One explanationfor these results is that the main role of the integrins during normalangiogenesis is to act as a “brake” and to prune unwanted new vessels byactivating cell death (apoptosis). Inhibitors that inactivate theintegrin and thus disrupt cell migration that relies on these integrinshave the additional effect of enhancing the apoptotic signal emanatingfrom the integrins, leading to endothelial cell death. This deathmechanism is lacking in mice in which the integrin gene is disrupted,and this may account for the enhanced angiogenesis seen in such knockoutmice.

A peptide representing the active site in the extracellular domain ofSdc1 (an inhibitory activity referred to as “synstatin”) competitivelydisrupts this positive regulation and blocks biological processes inwhich these two integrins participate, including angiogenesis. There area number of anti-angiogenic compounds that have been described and manyare in clinical trials. Some are generated in vivo, often by proteolysisof native matrix components, giving rise to angiostatin (O'Reilly etal., 1994), endostatin (O'Reilly et al., 1997), canstatin (Kamphaus etal., 2000), arresten (Colorado et al., 2000) and tumstatin (Maeshima etal., 2000). Tumstatin is of interest as it is derived from the α3 chainof collagen IV and the active site within tumstatin targets the αvβ3integrin. In light of the inventors' observations with regard to theactivity of synstatin, the fact that Tumstatin inhibits theproliferation of endothelial cells and is a highly effective inhibitorin angiogenesis assays is not surprising.

II. α_(v)β₃ and α_(v)β₅ Integrins

The α_(v)β₃ and α_(v)β₅ are closely related integrins that areupregulated during disease processes. The α_(v)β₃ integrin is a keyregulator of adhesion and signaling in numerous biological processes,including tumor cell migration and metastasis, and angiogenesis. Theactivated form of this integrin participates in arrest of tumor cells inthe blood stream (Pilch et al., 2002), enhancing their extravasation totarget tissues, especially bone, where the activated integrin hasfurther roles in tumor cell proliferation and survival (Brooks et al.,1994; Petitclerc et al., 1999; Eliceiri, 2001). In endothelial cellsforming new blood vessels, the active integrin is linked not only toadhesion-dependent processes but also to signaling in response to FGF-2(Eliceiri et al., 1998; Hood et al., 2003).

Although α_(v)β₃ integrin expression in mammary epithelium is low,activated α_(v)β₃ is expressed on most, if not all, successful mammarycarcinoma metastases (Liapis et al., 1996; Felding-Habermann et al.,2001). The inventors have reported previously that α_(v)β₃ integrin onMDA-MB-231 mammary carcinoma cells appears to be functionally linked toSdc-1; the cells spread when adherent to an artificial substratumcomprised solely of Sdc1-specific antibody, and although this spreadingoccurs in the absence of an α_(v)β₃ ligand, the spreading requiresactivated α_(v)β₃ integrin (Beauvais and Rapraeger, 2003). This findingsuggests that even on a native ECM, anchorage of Sdc-1 to the matrix mayserve as an important regulator of α_(v)β₃ integrin activation andsignaling.

Although classically defined as a vitronectin (VN) receptor, α_(v)β₃ ispromiscuous and binds many ECM components including fibronectin (FN),fibrinogen, von Willebrand Factor, proteolysed fragments of collagen(COL), laminin (LN), osteopontin, and others (van der Flier andSonnenberg, 2001). Mechanisms leading to activation of this integrin arecomplex, including proteolytic cleavage (Ratnikov et al., 2002),conformational changes (affinity modulation), and clustering (aviditymodulation; Carman and Springer, 2003). Activation is regulated by“inside-out” signals from the cell interior and is stabilized by ligandinteractions that trigger “outside-in” signaling (Giancotti andRuoslahti, 1999). Cell surface receptors known to modulate α_(v)β₃activity include CD87/uPAR and CD47/IAP, which associate with the β₃integrin subunit via their extracellular domains (Lindberg et al., 1996;Xue et al., 1997) and may also regulate α_(v)β₃ function indirectly viaa pertussis toxin-sensitive G-protein signaling pathway (Gao et al.,1996; Degryse et al., 2001).

The α_(v)β₅ integrin is a close relative of the α_(v)β₃ integrin. Theα_(v)β₅ integrin is expressed on a variety of tissues and cell types,including endothelia, epithelia and fibroblasts (Felding-Habermann andCheresh, 1993; Pasqualini et al., 1993). It is closely related to theα_(v)β₃ integrin (56.1% identity and 83.5% homology between the twointegrin β-subunits) but is distinguished from the α_(v)β₃ by divergentsites near its ligand-binding domain and within the C-terminus of itscytoplasmic domain (McLean et al., 1990). It has a role in matrixadhesion to VN, FN, SPARC and bone sialoprotein (Plow et al., 2000) andis implicated in the invasion of gliomas and metastatic carcinoma cells(Brooks et al., 1997; Jones et al., 1997; Tonn et al., 1998), the latterespecially to bone (De et al., 2003). A second major role is inendocytosis, including endocytosis of VN (Memmo and McKeown-Longo, 1998;Panetti et al., 1995), the engulfment of apoptotic cells by phagocytes(Albert et al., 2000), and participation in the internalization of shedouter rod segments in the retinal pigmented epithelium (Finnemann,2003a; Finnemann, 2003b; Hall et al., 2003). A third major role is ingrowth factor-induced angiogenesis, where cooperative signaling by theα_(v)β₅ integrin and growth factors regulates endothelial cellproliferation and survival. Angiogenesis promoted by VEGF and TGFα inhuman umbilical vein endothelial cells relies on co-signaling with theα_(v)β₅ integrin, whereas FGF-2 and tumor necrosis factor-α collaboratewith the α_(v)β₃ integrin (Eliceiri and Cheresh, 1999; Friedlander etal., 1995).

III. Syndecans

A. The Syndecan Family

Cell surface adhesion receptors physically bind cells to theirextracellular matrix (ECM) and couple such interactions to intracellularsignaling mechanisms which influence gene expression, cell morphology,motility, growth, differentiation and survival (Roskelley et al., 1995;Miranti and Brugge, 2002). Many ECM ligands contain closely spacedproteoglycan- and integrin-binding domains, indicating that themolecular mechanisms by which cells recognize and interact with theirextracellular milieu may involve the formation of signaling complexescontaining both proteoglycans and integrins. Consequentially, these twotypes of receptors may act in concert to modulate cell adhesion andmigration. While the role of integrins in cell adhesion and signaling iswell established, the role of heparan sulfate proteoglycans (HSPGs) isnot well characterized.

The vertebrate syndecans are a family of four transmembrane HSPGs.Endowed by their heparan sulfate (HS) chains, syndecans bind a varietyof ECM ligands, including fibronectin (FN), laminin (LN), tenascin,thrombospondin (TSP), vitronectin (VN) and the fibrillar collagens (COL)(Bernfield et al., 1999). While the syndecan HS chains are essential formatrix binding, less is known about the role of syndecan core proteinsin cell adhesion signaling, although the core protein can affect ligandbinding interactions, as well as occupancy induced signaling (Rapraegerand Ott, 1998; Rapraeger, 2000).

The syndecans display a high degree of conservation within their coreproteins both across species and across family members. Like theintegrins, the syndecans lack intrinsic signaling activity. Their shortcytoplasmic tails (ca. 30 aa) consist of three regions, two of which areconserved amongst the four syndecans (C1 and C2) and which flank anintervening variable (V) region. Proteins known to interact with theseconserved domains are believed to link syndecan ligand bindinginteractions to the transduction of intracellular signals (Couchman etal., 2001). Each family member is uniquely defined by its ectodomainsand the V-regions of its cytoplasmic tail. Divergence within theseregions is believed to confer separate and distinct functions to eachindividual family member. Distinct roles for the V-regions of Sdc-2 and-4 in matrix assembly and focal adhesion formation respectively havebeen described (Klass et al., 2000; Woods and Couchman, 2001); however,specific functions for the syndecan ectodomains are almost whollyunknown with the noted exception of Sdc-1 and -4 which contain bindingsites for as yet unidentified cell surface receptor(s) (McFall andRapraeger, 1997; McFall and Rapraeger, 1998).

B. Syndecan Function in Cell Adhesion and Spreading

Current evidence suggests that the syndecan core proteins participate inadhesion-mediated signaling in collaboration with co-receptors at thecell surface. One example is Sdc-4 in focal adhesion and stress fiberformation, which requires both Sdc-4 and integrin engagement whereasneither is sufficient alone (Woods et al., 1986; Izzard et al., 1986;Streeter and Rees, 1987; Singer et al., 1987). The requirement for Sdc-4ligation can be overcome by treatment with phorbol esters (Woods andCouchman, 1994) or lysophosphatidic acid (LPA) (Saoncella et al., 1999)implicating PKC and RhoA in Sdc-4 signaling. While the mechanism bywhich Sdc-4 contributes to RhoA activation is not clear, it is knownthat Sdc-4 interacts directly with PKCα as well as phosphatidyl inositol4,5 bisphosphate (PIP2) via its cytoplasmic tail and these interactionspotentiate PKCα activity (Oh et al., 1997a; Oh et al., 1997b; Oh et al.,1998; Baciu and Goetinck, 1995).

While the mechanism by which Sdc-1 signals is not clear, there is ampleevidence implicating a signaling role for this receptor as well. Ectopicexpression of Sdc-1 in Schwann cells enhances cell spreading andpromotes the formation of focal adhesions (Hansen et al., 1994) andactin stress fibers (Carey et al., 1994a); similar morphological changesoccur when Sdc-1 is co-clustered with antibodies (Carey et al., 1994b).This response requires the cytoplasmic domain, since clustering of atruncated core protein did not induce reorganization of thecytoskeleton. Expression of Sdc-1 in human ARH-77 leukemia cells orhepatocellular carcinoma cells inhibits invasion of cells into COLmatrices (Liu et al., 1998; Ohtake et al., 1999). ARH-77 cellsexpressing a chimera comprised of the Sdc-1 ectodomain fused to theglycosyl-phosphatidyl inositol (GPI) tail of glypican-1 also fail toinvade a COL matrix demonstrating that Sdc-1's anti-invasive activityresides in its extracellular domain. In similar studies, Raji humanlymphoblastoid cells transfected with mouse Sdc-1 (Raji-S1) spread onTSP, FN and antibodies directed against the Sdc-1 ectodomain (Lebakkenand Rapraeger, 1996). This spreading is unaffected by truncation of thecytoplasmic domain, indicating that the Sdc-1 core protein interactswith and cooperatively signals through an associated transmembranesignaling partner. Analogous features have also been observed for Sdc-2(Granes et al., 1999) and Sdc-4 (Yamashita et al., 1999).

Potential syndecan signaling partners include cell surface integrins.Iba et al. (2000) demonstrated that mesenchymal cells when seeded on anHS-specific ligand, the cysteine rich domain of a disintegrin andmetalloprotease, ADAM-12/Meltrin α (rADAM12-cys), will spread in amanner that requires cooperate signaling of both syndecans and β₁integrins. These results imply that syndecan(s) can trigger signalingcascades required for cell spreading either by exposing a crypticbinding site for β₁ integrins, as has been proposed for FN (Khan et al.,1988), or by modulating the activation state of β₁ integrins.Interestingly, colon carcinoma cells attach but fail to spread onaADAM12-cys. However, exogenous stimulation of β₁ integrins with Mn²⁺ orβ₁ integrin function activating antibody, mAb 12G10, induced cellspreading, suggesting a mechanism whereby the syndecan activates β₁integrins is blocked in transformed cells.

C. Angiogenesis

The formation of new blood vessels, called angiogenesis, which occurs innormal development as well as in disease states, relies on inducingproliferation and migration of endothelial cells, and in controlling thesurvival or apoptosis of the cells to control the architecture of thenew vessels (vascular pruning). The αvβ₃ integrin has important roles inall three of these steps.

FGF and VEGF, two growth factors that are often released by tumors,cause endothelial cells to undergo angiogenesis. Blood vessels in thevicinity of the tumor respond to VEGF by becoming leaky (thus thealternate name “vascular permeability factor”) allowing fibronectin,vitronectin and fibrinogen in the blood to infiltrate the surroundingmatrix. These matrix ligands are critical adhesion and activationligands for the αvβ3 and αvβ5 integrins, which have roles in thechemotactic migration of the endothelial cells and in the survival ofthe cells during vessel pruning. A second response to the growthfactors, particularly FGF, is the activation of a neovessel developmentprogram that relies on Hox master genes (Boudreau et al., 1997; Myers etal., 2000; 2002). HoxD3 is initially expressed and controls a family ofgenes that are necessary for the initial migration process, includingupregulation of the αvβ3 integrin, MMPs and uPAR (Boudreau et al.,1997). Expression of HoxD3 is followed by HoxB3 that regulates themorphogenesis leading to formation of small vessels (Myers et al.,2000), and finally by the HoxD10 gene, which restores the maturephenotype of the cells (Myers et al., 2002); it is HoxD10 that isexpressed in resting, stable blood vessels in vivo.

The αvβ₃ integrin is important not only during endothelial cellmigration, but is an important player in the survival of the endothelialcells. Although endothelial cells in mature vessels are not readilysusceptible to apoptosis, angiogenic cells that are induced by growthfactors rely upon the continued presence of these factors for survival.This is shown experimentally by inducing angiogenesis with VEGF andcausing apoptosis by its withdrawal, or in vivo when the developingovarian follicle induces angiogenesis by release of VEGF and the newlyformed bloods vessels regress upon ovulation as the source of VEGF islost. Endothelial cells responding to VEGF have been shown to bedependent on signaling from the αvβ5 integrin in order to block thisapoptotic process (Brooks et al., 1994; Friedlander et al., 1995). Thus,inhibiting the integrin using anti-integrin antibodies will induceapoptosis of the endothelial cells responding to VEGF. Similarly,endothelial cells responding to fibroblast growth factor (FGF) aresusceptible to apoptosis unless there is coordinate signaling from theαvβ5 integrin.

There are a number of anti-angiogenic compounds that have been describedand many are in clinical trials. Some are generated in vivo, potentiallythe proteolysis of native matrix components, giving rise to angiostatin(O'Reilly et al., 1994), endostatin (O'Reilly et al., 1997), canstatin(Kamphaus et al., 2000), arresten (Colorado et al., 2000) and tumstatin(Maeshima et al., 2000). Tumstatin is of interest as it is derived fromthe α3 chain of Collagen IV and the active site within tumstatin is apeptide binding site that targets the αvβ₃ integrin, although it appearsdistinct from the RDG binding site of the integrin. Tumstatin inhibitsthe proliferation of endothelial cells and is a highly effectiveinhibitor in angiogenesis assays. As this invention describes theregulation of the αvβ₃ integrin by Sdc-1 in mammary carcinoma cells andin endothelial cells, and this dependence can be blocked by solubleS1ED, it is hypothesized that this inhibitor would be an effectiveinhibitor of angiogenesis as well. Perhaps more intriguing is itsactivity only against the endothelial cells that express Sdc-1. Althoughthere is not much information available, the information to dateindicates that resting endothelial cells lining adult blood vessels donot express Sdc-1. In contrast, expression of Sdc-1 is turned on whenthe cells are activated to undergo angiogenesis, such as occurs normallyin wounding, or occurs in abnormal conditions such as diabeticretinopathy, restenosis following blood catheter injury to bloodvessels, or tumor angiogenesis. Thus, it is intriguing that targetingthe Sdc-1 regulation of the αvβ3 integrin can provide not only anadditional opportunity for drug discovery, but the drug may be mostefficacious during angiogenesis itself.

There has not been a concerted examination of Sdc-1 expression invascular endothelium. Most reports suggest that it is expressed poorlyor not at all on resting, mature vascular endothelium that lines bloodvessels. However, there are reports that suggest it is expressed onactivated endothelial cells participating in angiogenesis in the woundedskin (Elenius et al., 1991; Gallo et al., 1996). Sdc-1 is not expressedin endothelial cells lining the rabbit aorta, but expression isupregulated following balloon catheter injury and persists for up to 12weeks following injury. There is a report that Sdc-1 is upregulated in asubset of vessels during tumor angiogenesis (Gotte et al., 2002). Thesestudies strongly suggest that Sdc-1 becomes expressed on activated cellsresponding to injury or growth factors. Cultured cells, such as humanaortic and human umbilical vein endothelial cells show expression atboth the protein and mRNA level, although expression in human umbilicalvein endothelial cells is low (Mertens et al., 1992). However, theexpression patterns described may be dependent on the growth factors andsupplements, such as brain extract, added to the culture medium.

D. Syndecan-1

Syndecan-1 is highly expressed at the basolateral surface of epithelialcells where it is thought to interact with the actin cytoskeleton and tomodulate cell adhesion and growth factor signaling (Bernfield et al.,1999; Rapraeger et al., 1986; Kim et al., 1994; Sanderson and Bernfield,1988). In experimental studies of malignant transformation, Sdc-1expression is associated with the maintenance of epithelial morphology,anchorage-dependent growth and inhibition of invasiveness. Alterationsin syndecan expression during development (Sun et al., 1998) and intransformed epithelial (Inki and Jalkanen, 1996; Bayer-Garner et al.,2001) are associated with an epithelial-mesenchymal transformation withattendant alterations in cell morphology, motility, growth anddifferentiation. Transfection of epithelial cells with anti-sense mRNAfor Sdc-1 or downregulation of Sdc-1 expression by androgen-inducedtransformation results in an epithelial to mesenchymal transformationand increased invasion (Leppa et al., 1992; Kato et al., 1995; Leppa etal., 1991). The loss of E-cadherin under these circumstances has longsuggested a coordinate regulation of Sdc-1 and E-cadherin expression(Sun et al., 1998; Leppa et al., 1996). These studies, as well asothers, indicate that there appears to be a threshold requirement forsyndecan expression to elicit its biological activity. Syndecan-1 isdownregulated in a number of epithelial cancers and in pre-malignantlesions of the oral mucosa (Soukka et al., 2000) and uterine cervix(Inki et al., 1994; Rintala et al., 1999; Nakanishi et al., 1999), andits loss may be an early genetic event contributing to tumor progression(Sanderson, 2001; Numa et al., 2002; Hirabayashi et al., 1998). Loss ofSdc-1 correlates with a reduced survival in squamous cell carcinoma ofthe head, neck and lung (Anttonen et al., 1999; Inki et al., 1994;Nakaerts et al., 1997), laryngeal cancer (Pulkkinen et al., 1997;Klatka, 2002), malignant mesothelioma (Kumar-Singh et al., 1998) andmultiple myeloma (Sanderson and Borset, 2002) and a high metastaticpotential in hepatocellular and colorectal carcinomas (Matsumoto et al.,1997; Fujiya et al., 2001; Levy et al., 1997; Levy et al., 1996).Downregulation of Sdc-2 and -4 expression has also been observed incertain human carcinomas (Nakaerts et al., 1997; Park et al., 2002;Mundhenke et al., 2002; Crescimanno et al., 1999), but the functionalconsequences of these alterations in expression are less clear.

In contrast to the general notion that the syndecan may be an inhibitorof carcinogenesis, Sdc-1 also demonstrates tumor promoter function.Syndecan-1 supplements Wnt-1 induced tumorigenesis of the mouse mammarygland (Alexander et al., 2000) and promotes the formation of metastasesin mouse lung squamous carcinoma cells (Hirabayashi et al., 1998).Enhanced Sdc-1 expression has also been observed in pancreatic (Conejoet al., 2000), gastric (Wiksten et al., 2001) and breast (Burbach etal., 2003; Stanley et al., 1999; Barbareschi et al., 2003) carcinomasand this overexpression correlates with increased tumor aggressivenessand poor clinical prognosis. This duality in the role of Sdc-1 intumorigenesis may reflect tissue and/or tumor stage-specific function,or reflect the multiple functions of this PG.

Sanderson was the first to demonstrate a role for Sdc-1 in tumor cellmigration by examining the invasion of myeloma cells into collagen gels(Liu et al., 1998). Ectopic expression of Sdc-1 in syndecan-deficientmyeloma cells had the striking effect of curtailing invasion, whereasthe expression of other cell surface heparan sulfate PGs (e.g.,glypican) was without effect. Using chimeras derived from these twoproteins, Sanderson showed that the activity of the syndecan ispreserved when its ectodomain alone is expressed as aglycosyl-phosphatidylinositol (GPI)-linked protein at the cell surface.Although clearly responsible for binding the collagen matrix via itsattached heparan sulfate chains, the anti-invasive activity of thesyndecan requires yet an additional interaction that traces to a site inthe extracellular domain of the core protein itself. The mechanism bywhich the ectodomain site influences the invasion of the myeloma cellsis unknown, but its interaction with other cell surface receptors in a“co-receptor” role is one possibility. More recently, ectopic expressionof Sdc-1 has also been shown to curtail the invasion of hepatocellularcarcinoma cells into a collagen matrix (Ohtake et al., 1999).

E. Proteins and Peptides

Syndecan-1 peptides and polypeptides of the present invention willgenerally comprise molecules of 5 to about 240 residues in length, andmay have the sequence of SEQ ID NO:1 or SEQ ID NO:4 or SEQ ID NO:8 orSEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:13 or SEQ ID NO:21 or SEQ IDNO:23 or SEQ ID NO:28. A particular length may be 234 residues, 34residues, 32 residues, less than 30 residues, less than 25 residues,less than 20 residues, less than 15 residues, or less than 14 residues,including 5, 6, 7, 8, 9, 10, 11, 12, or 13 residues. In otherembodiments, the peptides or proteins may be from SEQ ID NOS:2, 4, 8, 9,13, 21, 23, or 28 and may thus comprise 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 49, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, or 240 consecutive residues of that sequence.Alternatively, the peptides may have 90%, 95%, or more identity with theSEQ ID NOs identified herein. It is further contemplated that anyembodiment involving a mouse sequence discussed herein may beimplemented with the corresponding human sequence. Such human peptidesand nucleic acids encoding such peptides are specifically contemplatedas part of the invention.

The peptides or proteins may be generated synthetically or byrecombinant techniques, and are purified according to known methods,such as precipitation (e.g., ammonium sulfate), HPLC, ion exchangechromatography, affinity chromatography (including immunoaffinitychromatography) or various size separations (sedimentation, gelelectrophoresis, gel filtration).

Accordingly, sequences that have between about 70% and about 80%,between about 81% and about 90%, between about 91% and 95%, or about 96,about 97%, 98% or about 99% of amino acids that are identical orfunctionally equivalent to the amino acids of SEQ ID NOS:2, 4, 8, 9 or10, will be sequences that are “essentially as set forth in SEQ IDNOS:2, 4, 8, 9 or 10.” Peptides and polypeptides of the invention may beas essentially as set forth in SEQ ID NOS:2, 4, 8, 9 or 10.

i. Substitutional Variants

It also is contemplated in the present invention that variants oranalogs of syndecan-1 peptides or proteins may also inhibit tumorgrowth. Polypeptide sequence variants of syndecan-1, primarily makingconservative amino acid substitutions to SEQ ID NOS:2, 4, 8, 9 or 10,may provide improved compositions. Substitutional variants typicallycontain the exchange of one amino acid for another at one or more siteswithin the protein, and may be designed to modulate one or moreproperties of the polypeptide, such as stability against proteolyticcleavage, without the loss of other functions or properties.Substitutions of this kind preferably are conservative, that is, oneamino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

The following is a discussion based upon changing of the amino acids ofa peptide or protein to create an equivalent, or even an improved,second-generation molecule. For example, certain amino acids may besubstituted for other amino acids in a protein structure withoutappreciable loss of interactive binding capacity with structures suchas, for example, antigen-binding regions of antibodies or binding siteson substrate molecules. Since it is the interactive capacity and natureof a peptide that defines that peptide's biological functional activity,certain amino acid substitutions can be made in a protein sequence, andits underlying DNA coding sequence, and nevertheless obtain a peptidewith like properties. It is thus contemplated by the inventors thatvarious changes may be made in syndecan-1 amino acid sequences and inthe DNA sequences coding the peptide without appreciable loss of theirbiological utility or activity, as discussed below.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant peptide, which in turn defines theinteraction of the peptide with other molecules.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a peptide with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine*−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics are peptidecontaining molecules that mimic elements of protein secondary structure(Johnson et al, 1993). The underlying rationale behind the use ofpeptide mimetics is that the peptide backbone of proteins exists chieflyto orient amino acid side chains in such a way as to facilitatemolecular interactions, such as those of antibody and antigen. A peptidemimetic is expected to permit molecular interactions similar to thenatural molecule. These principles may be used, in conjunction with theprinciples outline above, to engineer second generation molecules havingmany of the natural properties of syndecan-1, but with altered and evenimproved characteristics.

ii. Altered Amino Acids

The present invention may employ peptides that comprise modified,non-natural and/or unusual amino acids. A table of exemplary, but notlimiting, modified, non-natural and/or unusual amino acids is providedherein below. Chemical synthesis may be employed to incorporate suchamino acids into the peptides of interest. TABLE 1 Modified, Non-Naturaland Unusual Amino Acids Abbr. Amino Acid Aad 2-Aminoadipic acid BAad3-Aminoadipic acid BAla beta-alanine, beta-Amino-propionic acid Abu2-Aminobutyric acid 4Abu 4-Aminobutyric acid, piperidinic acid Acp6-Aminocaproic acid Ahe 2-Aminoheptanoic acid Aib 2-Aminoisobutyric acidBAib 3-Aminoisobutyric acid Apm 2-Aminopimelic acid Dbu2,4-Diaminobutyric acid Des Desmosine Dpm 2,2′-Diaminopimelic acid Dpr2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsn N-EthylasparagineHyl Hydroxylysine Ahyl allo-Hydroxylysine 3Hyp 3-Hydroxyproline 4Hyp4-Hydroxyproline Ide Isodesmosine Aile allo-Isoleucine MeGlyN-Methylglycine, sarcosine MeIle N-Methylisoleucine MeLys6-N-Methyllysine MeVal N-Methylvaline Nva Norvaline Nle Norleucine OrnOrnithine

iii. Mimetics

In addition to the variants discussed above, the present inventors alsocontemplate that structurally similar compounds may be formulated tomimic the key portions of peptide or polypeptides of the presentinvention. Such compounds, which may be termed peptidomimetics, may beused in the same manner as the peptides of the invention and, hence,also are functional equivalents.

Certain mimetics that mimic elements of protein secondary and tertiarystructure are described in Johnson et al. (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and/or antigen. A peptide mimetic is thus designed to permitmolecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focusedon mimetics of β-turns within proteins, which are known to be highlyantigenic. Likely β-turn structure within a polypeptide can be predictedby computer-based algorithms, as discussed herein. Once the componentamino acids of the turn are determined, mimetics can be constructed toachieve a similar spatial orientation of the essential elements of theamino acid side chains.

Other approaches have focused on the use of small,multidisulfide-containing proteins as attractive structural templatesfor producing biologically active conformations that mimic the bindingsites of large proteins (Vita et al., 1998). A structural motif thatappears to be evolutionarily conserved in certain toxins is small (30-40amino acids), stable, and high permissive for mutation. This motif iscomposed of a beta sheet and an alpha helix bridged in the interior coreby three disulfides. Beta II turns have been mimicked successfully usingcyclic L-pentapeptides and those with D-amino acids (Weisshoff et al.,1999). Also, Johannesson et al. (1999) report on bicyclic tripeptideswith reverse turn inducing properties.

Methods for generating specific structures have been disclosed in theart. For example, alpha-helix mimetics are disclosed in U.S. Pat. Nos.5,446,128; 5,710,245; 5,840,833; and 5,859,184. These structures, whichrender the peptide or protein more thermally stable, also increaseresistance to proteolytic degradation. Six, seven, eleven, twelve,thirteen and fourteen membered ring structures are disclosed.

Methods for generating conformationally restricted beta turns and betabulges are described, for example, in U.S. Pat. Nos. 5,440,013;5,618,914; and 5,670,155. Beta-turns permit changed side substituentswithout having changes in corresponding backbone conformation, and haveappropriate termini for incorporation into peptides by standardsynthesis procedures. Other types of mimetic turns include reverse andgamma turns. Reverse turn mimetics are disclosed in U.S. Pat. Nos.5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S.Pat. Nos. 5,672,681 and 5,674,976.

iv. D Amino Acids

In another form, the present invention contemplates use of variants thatcomprise various portions of an syndecan-1 peptide or protein in reverseorder of SEQ ID NOS:2, 4, 8, 9 or 10, using D amino acids, stereoisomersof natural amino acids which are in the L-form.

v. Peptide Synthesis

Syndecan-1 and related peptides may be generated synthetically for usein various embodiments of the present invention. Because of theirrelatively small size, the peptides of the invention can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart and Young (1984); Tam et al. (1983); Merrifield (1986); Baranyand Merrifield (1979), each incorporated herein by reference. Shortpeptide sequences, or libraries of overlapping peptides, usually fromabout 5 up to about 34 to 40 amino acids, which correspond to theselected regions described herein, can be readily synthesized and thenscreened in screening assays designed to identify reactive peptides.Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of the invention is insertedinto an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression.

It may be desirable to purify syndecan-1 variants, peptide-mimics oranalogs thereof. Protein purification techniques are well known to thoseof skill in the art. These techniques involve, at one level, the crudefractionation of the cellular milieu to polypeptide and non-polypeptidefractions. Having separated the polypeptide from other proteins, thepolypeptide of interest may be further purified using chromatographicand electrophoretic techniques to achieve partial or completepurification (or purification to homogeneity). Analytical methodsparticularly suited to the preparation of a pure peptide areion-exchange chromatography, exclusion chromatography; polyacrylamidegel electrophoresis; isoelectric focusing. A particularly efficientmethod of purifying peptides is fast protein liquid chromatography oreven HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

IV. Anti-Idiotypic Antibodies

The present invention also provide antibodies that mimic the syndecan-1peptides and proteins described herein. These antibodies are created byfirst preparing an antibody against a syndecan-1 peptide or protein andthen preparing a second antibody, called an anti-idiotypic antibody,against the idiotype of the first antibody. As a mirror image of amirror image, the binding site of anti-idiotype would be expected to bean analog of the original antigen. As used herein, the term “antibody”is intended to refer broadly to any immunologic binding agent such asIgG, IgM, IgA, IgD and IgE. Generally, IgG and/or IgM are preferredbecause they are the most common antibodies in the physiologicalsituation and because they are most easily made in a laboratory setting.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)₂, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibodies and antibody-based constructs and fragments are well known inthe art (see, e.g., Harlow et al., 1988; and U.S. Pat. No. 4,196,265each incorporated herein by reference).

V. Nucleic Acid Segments Encoding Syndecan Peptides and Proteins

The present invention concerns nucleic acid segments, isolatable fromcells, that are free from total genomic DNA and that are capable ofexpressing all or part of a protein or polypeptide such as syndecan-1 orthe syndecan-1 peptide or protein of SEQ ID NOS:2, 4, 8, 9, 10, 21, 23,or 28. The nucleic acid may encode a peptide or polypeptide containingall or part of the syndecan-1 amino acid sequence.

As used herein, the term “nucleic acid segment” refers to a DNA moleculethat has been isolated free of total genomic DNA of a particularspecies. Therefore, a nucleic acid segment encoding a syndecan-1 refersto a nucleic acid segment that contains wild-type, polymorphic, ormutant polypeptide-coding sequences yet is isolated away from, orpurified free from, total mammalian or human genomic DNA. Includedwithin the term “nucleic acid segment” are a polypeptide(s), nucleicacid segments smaller than a polypeptide, and recombinant vectors,including, for example, plasmids, cosmids, phage, viruses, and the like.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

A nucleic acid segment encoding all or part of a native or modifiedpolypeptide may contain a contiguous nucleic acid sequence encoding allor a portion of such a polypeptide of the following lengths: about 10,about 20, about 30, about 40, about 50, about 60, about 70, about 80,about 90, or about 100, about 110, about 120, about 130, about 140,about 150, about 160, about 170, about 180, about 190, about 200, about210, about 220, about 230, about 240, about 250 or about 260nucleotides, nucleosides, or base pairs. Specifically contemplated are asegment encoding SEQ ID NO:2, a segment encoding the 34 residue peptideof SEQ ID NO:4, a segment encoding residues 18-251 residues of SEQ IDNO:2 (SEQ ID NO:8), a segment encoding residues 18-310 of SEQ ID NO:2(SEQ ID NO:9) or residues 82-130 of SEQ ID NO:2 (SEQ ID NO:10).Additionally, nucleic acids encoding a peptide consisting of orcomprising the amino acid sequence of SEQ ID NOs:1, 21, 23, or 28.

The nucleic acid segments used in the present invention, regardless ofthe length of the coding sequence itself, may be combined with othernucleic acid sequences, such as promoters, enhancers polyadenylationsignals, origin of replication, and a selectable marker gene, as well asother coding segments, and the like (all as are known to those ofordinary skill in the art), such that their overall length may varyconsiderably.

The term oligonucleotide refers to at least one molecule of betweenabout 3 and about 100 nucleobases in length. These definitions generallyrefer to at least one single-stranded molecule, but in specificembodiments will also encompass at least one additional strand that ispartially, substantially or fully complementary to the at least onesingle-stranded molecule. Thus, a nucleic acid may encompass at leastone double-stranded molecule or at least one triple-stranded moleculethat comprises one or more complementary strand(s) or “complement(s)” ofa particular sequence comprising a strand of the molecule.

It is contemplated that the nucleic acid constructs of the presentinvention may encode full-length polypeptide from any source or encode atruncated version of the polypeptide, for example a truncated syndecan-1polypeptide, such that the transcript of the coding region representsthe truncated version. The truncated transcript may then be translatedinto a truncated protein. Alternatively, a nucleic acid sequence mayencode a full-length polypeptide sequence with additional heterologouscoding sequences, for example to allow for purification of thepolypeptide, transport, secretion, post-translational modification, orfor therapeutic benefits such as targeting or efficacy.

It is contemplated that the nucleic acid constructs of the presentinvention may regulate gene expression of an immunogenic polypeptide. Anucleic acid segment may regulate the expression of a full-lengthpolypeptide sequence with additional heterologous coding sequences, forexample to allow for therapeutic benefits such as targeting or efficacy.

In a non-limiting example, one or more nucleic acid constructs may beprepared that include a contiguous stretch of nucleotides identical toor complementary to a particular gene, such as the human syndecan-1 gene(SEQ ID NO:1), or a fragment thereof (SEQ ID NO:3 or that encoding SEQID NOS:8 or 9 or 10). Such a nucleic acid construct may be at least 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000,4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 15,000, 20,000,30,000, 50,000, 100,000, 250,000, 500,000, 750,000, to at least1,000,000 nucleotides in length, as well as constructs of greater size,up to and including chromosomal sizes (including all intermediatelengths and intermediate ranges), given the advent of nucleic acidsconstructs such as a yeast artificial chromosome are known to those ofordinary skill in the art. It will be readily understood that“intermediate lengths” and “intermediate ranges,” as used herein, meansany length or range including or between the quoted values (i.e., allintegers including and between such values).

In certain other embodiments, the invention concerns isolated nucleicacid segments and recombinant vectors that include within their sequencea contiguous nucleic acid sequence from that shown in SEQ ID NO:1, 3 orencoding SEQ ID NOS: 8 or 9 or 10 or 21 or 23 or 28. This definition isused in the same sense as described above and means that the nucleicacid sequence substantially corresponds to a contiguous portion of thatshown in SEQ ID NO:1 or 3 or encoding SEQ ID NOS: 8 or 9 or 10 or 21 or23 or 28 and has relatively few codons that are not identical, orfunctionally equivalent, to the codons of SEQ ID NO:1, 3 or encoding SEQID NOS: 8, 9, 10, 21, 23, or 28. The term “functionally equivalentcodon” is used herein to refer to codons that encode the same aminoacid, such as the six codons for arginine or serine, and also refers tocodons that encode biologically equivalent amino acids, as is known tothose of skill in the art.

It also will be understood that this invention is not limited to theparticular nucleic acid sequence of SEQ ID NO:1 and the amino acidsequence of SEQ ID NO:2. Recombinant vectors and isolated DNA segmentsmay therefore variously include the syndecan-1-coding regionsthemselves, coding regions bearing selected alterations or modificationsin the basic coding region, or they may encode larger polypeptides thatnevertheless include syndecan-1-coding regions or may encodebiologically functional equivalent proteins or peptides that havevariant amino acids sequences.

The nucleic acid segments of the present invention encompassbiologically functional equivalent syndecan-1 proteins and peptides.Such sequences may arise as a consequence of codon redundancy andfunctional equivalency that are known to occur naturally within nucleicacid sequences and the proteins thus encoded. Alternatively,functionally equivalent proteins or peptides may be created via theapplication of recombinant DNA technology, in which changes in theprotein structure may be engineered, based on considerations of theproperties of the amino acids being exchanged. Changes designed by manmay be introduced through the application of site-directed mutagenesistechniques, e.g., to introduce improvements to the antigenicity of theprotein.

If desired, one also may prepare fusion proteins and peptides, e.g.,where the syndecan-1-coding regions are aligned within the sameexpression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins that may be purified by affinity chromatography and enzymelabel coding regions, respectively).

Encompassed by certain embodiments of the present invention are nucleicacid segments encoding relatively small peptides, such as, for example,peptides of from about 5 to about 40 amino acids in length, and morepreferably, of from about 10 to about 34 amino acids in length; and alsolarger polypeptides up to and including proteins corresponding to thefull-length sequence set forth in SEQ ID NO:2, the peptide of SEQ IDNO:4, the polypeptide of SEQ ID NOS:8 or 9, or the peptide of SEQ IDNOs:10, 21, 22, 23, or 28, or to specific nucleic acid fragments of SEQID NO:1, such as SEQ ID NO:3 and those encoding SEQ ID NOS: 8 or 9 or 10or 21, 23, or 28.

A. Promoters

The present invention may also involve expression of sdc-1 or relatedpeptide from a sdc-1-encoding nucleic acid. This requires the presenceof a promoter operably linked to the sdc-1-coding region. A promotergenerally comprises a nucleic acid sequence that functions to positionthe start site for RNA synthesis. A promoter may or may not be used inconjunction with an enhancer, which refers to a cis-acting regulatorysequence involved in the transcriptional activation of a nucleic acidsequence. In the present invention, a nucleic acid encoding a sdc-1comprises a promoter such as a tissue specific promoter, or aconstitutive promoter, or an inducible promoter.

A promoter in the context of the present invention may be one naturallyassociated with a gene or sequence, as may be obtained by isolating the5′ non-coding sequences located upstream of the coding segment and/orexon. Such a promoter can be referred to as “endogenous.” Similarly, anenhancer may be one naturally associated with a nucleic acid sequence,located either downstream or upstream of that sequence. Alternatively,certain advantages will be gained by positioning the coding nucleic acidsegment under the control of a recombinant or heterologous promoter orenhancer, which refers to a promoter or enhancer, that is not normallyassociated with a nucleic acid sequence in its natural environment. Suchpromoters or enhancers may include promoters or enhancers of othergenes, and promoters or enhancers isolated from any other prokaryotic,viral, or eukaryotic cell, and promoters or enhancers not “naturallyoccurring,” i.e., containing different elements of differenttranscriptional regulatory regions, and/or mutations that alterexpression. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. No.4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein byreference). Furthermore, it is contemplated the control sequences thatdirect transcription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,for example, see Sambrook et al. (2000), incorporated herein byreference.

The present invention also contemplates the use of tissue specificpromoters and inducible promoters. Other promoters that may be employedwith the present invention are constitutive and inducible promoters asare well known to those of skill in the art. Additionally anypromoter/enhancer combination (as per the Eukaryotic Promoter Data BaseEPDB) could also be used to drive expression of structural genesencoding oligosaccharide processing enzymes, protein folding accessoryproteins, selectable marker proteins or a heterologous protein ofinterest.

B. Origins of Replication/Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention. Polyadenylationsignals include the SV40 polyadenylation signal and the bovine growthhormone polyadenylation signal, known to function well in various targetcells. Polyadenylation may increase the stability of the transcript ormay facilitate cytoplasmic transport.

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

C. Delivery of Nucleic Acids

Two broad approaches have been used to employ vectors to deliver nucleicacids to cells, namely viral vectors and non-viral vectors. As bymethods described herein and as is known to the skilled artisan,expression vectors may be constructed to deliver nucleic acids segmentsencoding a syndecan-1 of the present invention to a organelle, cell,tissue, or a subject. Such vectors comprising a syndecan-1 may be usedin a variety of manner consistent with the invention, including inscreening assay and genetic immunization protocols.

A vector in the context of the present invention refers to a carriernucleic acid molecule into which a nucleic acid sequence of the presentinvention may be inserted for introduction into a cell and therebyreplicated. A nucleic acid sequence can be exogenous, which means thatit is foreign to the cell into which the vector is being introduced; orthat the sequence is homologous to a sequence in the cell but positionedwithin the host cell nucleic acid in which the sequence is ordinarilynot found. Vectors include plasmids; cosmids; viruses such asbacteriophage, animal viruses, and plant viruses; and artificialchromosomes (e.g., YACs); and synthetic vectors. One of ordinary skillin the art would be well equipped to construct any number of vectorsthrough standard recombinant techniques as described in Maniatis et al.,1990 and Ausubel et al., 1994, incorporated herein by reference.

Viral vectors may be derived from viruses known to those of skill in theart, for example, bacteriophage, animal and plant virus, including butnot limited to, adenovirus, vaccinia virus (Ridgeway, 1988; Baichwal andSugden, 1986; Coupar et al., 1988) adeno-associated virus (AAV)(Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984)retrovirus and herpesvirus and offer several features for use in genetransfer into various mammalian cells (Friedmann, 1989; Ridgeway, 1988;Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).One of skill in the art would be well equipped to construct a vectorthrough standard recombinant techniques as described in Sambrook et al.(2001), Maniatis et al. (1990) and Ausubel et al. (1994), incorporatedherein by reference. The present invention may also employ non-viralvectors.

An expression vector refers to any type of genetic construct comprisinga nucleic acid coding for a RNA capable of being transcribed. In thecontext of the present specification, expression vectors will typicallycomprise a nucleic acid segment encoding a syndecan-1 as describedherein. In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, as in the case of antisense molecules or ribozymesproduction. Expression vectors can contain a variety of controlsequences, which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell, and are described herein

Non-viral vectors, such as plasmids and cosmids, require suitable methodfor delivery into cells. Such methods include, but are not limited todirect delivery of DNA by: injection (U.S. Pat. Nos. 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610,5,589,466 and 5,580,859, each incorporated herein by reference),microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215,incorporated herein by reference); electroporation (U.S. Pat. No.5,384,253, incorporated herein by reference; Tur-Kaspa et al., 1986;Potter et al., 1984); calcium phosphate precipitation (Graham and VanDer Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); usingDEAE-dextran followed by polyethylene glycol (Gopal, 1985); direct sonicloading (Fechheimer et al., 1987); by liposome-mediated transfection(Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wonget al., 1980; Kaneda et al., 1989; Kato et al., 1991); receptor-mediatedtransfection (Wu and Wu, 1987; Wu and Wu, 1988); microprojectilebombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat.Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880,and each incorporated herein by reference); agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference);desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed. In certain embodiments, accelerationmethods are preferred and include, for example, microprojectilebombardment or inhalation methods.

Also in context of the present invention, topical delivery of a nucleicacid segment encoding a syndecan-1 to the skin may further comprisevesicles such as liposomes, niosomes and transferosomes therebyenhancing topical and transdermal delivery. Cationic lipids may also beused to deliver negatively charged nucleic acids. Sonophoresis orphonophoresis which involves the use of ultrasound to deliver thenucleic acid of interest, may also be employed for transdermal delivery.Ionotophoresis which consists of applying a low electric field for aperiod of time to the skin may also be applied in delivering the nucleicacid of interest to the skin.

VI. Pharmaceutical Formulations, Delivery, and Cancer Treatment Regimens

In particular embodiments of the present invention, a method oftreatment for cancer by the delivery of a Sdc-1 peptide or polypeptide(as described elsewhere in this document) is contemplated. Cancerscontemplated by the present invention include, but are not limited to,breast cancer, lung cancer, head and neck cancer, bladder cancer, bonecancer, bone marrow cancer, brain cancer, colon cancer, esophagealcancer, gastrointestinal cancer, gum cancer, kidney cancer, livercancer, nasopharynx cancer, ovarian cancer, prostate cancer, skincancer, stomach cancer, testis cancer, tongue cancer, or uterine cancer.In particular embodiments, carcinomas, myelomas, melanomas or gliomasmay be treated.

A variety of other non-cancer angiogenic diseases may also be treatedwith the Sdc-1 peptides or proteins of the present invention. Theseinclude angiogenesis leading to abnormalities of the vasculature(atherosclerosis and hemangiomas), of the eye (diabetic retinopathy andretinopathy of prematurity), the skin (pyogenic granulomas, psoriasis,warts, scar keloids, allergic edema, ulcers), the uterus and ovary(dysfunctional uterine bleeding, follicular cysts, endometriosis,pre-eclampsia) adipose tissue (obesity), bones and joints (Rheumatoidarthritis, osteophyte formation), and AIDS-related pathologies resultingfrom TAT protein of the human immunodeficiency virus (HIV) activatingthe avb3 integrin on endothelial cells (Carmeliet and Jain, 2000;Urbinati et al., 2005).

A. Administration

To inhibit angiogenesis in, e.g., cancer, one would generally contact acell or tissue that has or can promote or undergo angiogenesis, with aSdc-1 peptide or protein or an expression construct encoding a Sdc-1peptide or protein. The preferred method for the delivery of a peptideor an expression construct is via injection. Administration may beparenteral, intradermal, intramuscular, or intratumoral administration.Other administration routes include lavage, continuous perfusion,topical and oral administration and formulation. See U.S. Pat. Nos.5,543,158; 5,641,515; 5,399,363 (each specifically incorporated hereinby reference in its entirety). Injection of nucleic acid constructs ofthe present invention may be delivered by syringe or any other methodused for injection of a solution, as long as the expression constructcan pass through the particular gauge of needle required for injection.A needleless injection system (U.S. Pat. No. 5,846,233); or a syringesystem for use in gene therapy (U.S. Pat. No. 5,846,225), all asincorporated herein by reference, may be employed in the presentinvention.

B. Compositions and Formulations

Pharmaceutical forms suitable for injectable use include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions (U.S. Pat.No. 5,466,468, specifically incorporated herein by reference in itsentirety). In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. Composition(s) of absorptiondelay agents (aluminum monostearate and gelatin) may also be used. Itmust be stable under the conditions of manufacture and storage and mustbe preserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose (see forexample, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). These particular aqueous solutions areespecially suitable for subcutaneous, intramuscular, and intratumoraladministration. In this connection, sterile aqueous media that may beemployed will be known to those of skill in the art in light of thepresent disclosure. Variation in dosage will necessarily occur dependingon the condition of the subject being treated; the severity of thecondition, and will be determined by the person administering the dose.For human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts, include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids; or salts (formed with the freecarboxyl groups) derived from inorganic bases as is known to those ofordinary skill in the art.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” or“pharmacologically-acceptable” refers to molecular entities andcompositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art.

C. Combination Treatments

In the context of the present invention, it is contemplated that Sdc-1peptides, proteins or analogs thereof may be used in combination with anadditional therapeutic agent to more effectively treat a cancer or otherangiogenic diseases.

Additional therapeutic agents contemplated for use in combination withSdc-1 peptides, proteins or analogs thereof include, in the case ofcancers, traditional anti-cancer therapies. Anticancer agents mayinclude but are not limited to, radiotherapy, chemotherapy, genetherapy, hormonal therapy, surgery or immunotherapy that targetscancer/tumor cells.

To kill cells, induce cell-cycle arrest, inhibit migration, inhibitmetastasis, inhibit survival, inhibit proliferation, or otherwisereverse or reduce the malignant phenotype of cancer cells, using themethods and compositions of the present invention, one would generallycontact a cell with sdc-1 peptides, proteins or an analog thereof incombination with an additional therapeutic agent. These compositionswould be provided in a combined amount effective to inhibit cell growthand/or induce apoptosis in the cell.

For other angiogenic diseases, the combination therapy may includeadministration of a second anti-angiogenic therapy. This process mayinvolve contacting the cells with Sdc-1 peptides, proteins or analogsthereof in combination with an additional therapeutic agent or factor(s)at the same time. This may be achieved by contacting the cell with asingle composition or pharmacological formulation that includes bothagents, or by contacting the cell with two distinct compositions orformulations, at the same time, wherein one composition includes theSdc-1 peptides, proteins or derivatives thereof and the other includesthe additional agent.

Alternatively, treatment with Sdc-1 peptides, proteins or analogsthereof may precede or follow the additional agent treatment byintervals ranging from minutes to weeks. In embodiments where theadditional agent is applied separately to the cell, one would generallyensure that a significant period of time did not expire between the timeof each delivery, such that the agent would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one would contact the cell with both modalities withinabout 12-24 hr of each other and, more preferably, within about 6-12 hrof each other, with a delay time of only about 12 hr being mostpreferred. Thus, therapeutic levels of the drugs will be maintained. Insome situations, it may be desirable to extend the time period fortreatment significantly (for example, to reduce toxicity). Thus, severaldays (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8)may lapse between the respective administrations.

It also is conceivable that more than one administration of eithersyndecan-1 peptides or analogs thereof in combination with an additionalanticancer agent will be desired. Various combinations may be employed,where Sdc-1 peptide, protein or an analog thereof is “A” and theadditional therapeutic agent is “B”, as exemplified below: A/B/A B/A/BB/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/ B A/A/B/B A/B/A/B A/B/B/A B/B/A/AB/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/ A/B/BB/B/A/BOther combinations are contemplated.

VIII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods

Cells. Human dermal microvascular endothelial cells (HMEC-1) and humanaortic endothelial cells (HAEC) were grown in endothelial cell growthmedium supplemented with 10% FBS serum. B82L mouse fibroblasts and humanmammary carcinoma MDA-MB-231 cells were cultured in Dulbecco's ModifiedEagle's medium supplemented with 10% FBS (Beauvais and Rapraeger, 2003;Beauvais et al., 2004; McQuade et al., 2006).

Recombinant mScd-1ED and synstatin₈₂₋₁₃₀ inhibitors. A GST fusionprotein consisting of the mScd-1ED (amino acids 18-251) was used as acompetitor in cell adhesion studies (Beauvais and Rapraeger, 2003;McFall and Rapraeger, 1998). The protein was expressed in bacteria andpurified on a glutathione affinity column as described in previouspublications (Beauvais and Rapraeger, 2003; McFall and Rapraeger, 1998).Synstatin₈₂₋₁₃₀ peptide represents amino acids 82-130 of mouse Sdc-1.The peptide was synthesized and purified to 75% purity by GenScriptCorporation (Piscataway, N.J.).

Cell spreading assays. Cell adhesion and spreading assays were performedwith modification to the previous procedures (Lebakken and Rapraeger,1996. Briefly, human plasma vitronectin or fibronectin at 10-30 μg/mlwere applied to nitrocellulose-coated ten well slides (Erie Scientific)and incubated for 1-2 h at 37° C. Slides were blocked with 1.0%heat-denatured bovine serum albumin for a minimum of 30 min at 37° C.Cells were detached from the substratum using 5 mM EDTA in Tris-bufferedsaline and resuspended in HEPES-buffered culture medium containing 0.1%heat-denatured BSA. Cells were plated at a density of 15,000 cells perwell and allowed to attach and spread for 2 h prior to fixation in 4%paraformaldehyde in CMF-PBS. For fluorescence microscopy, fixed cellswere permeabilized in 0.2% Triton X-100, labeled withrhodamine-conjugated phalloidin and analyzed using fluorescencemicroscopy. For testing inhibition by recombinant mScd-1ED andsynstatin₈₂₋₁₃₀, cells were pre-incubated 10 min before plating in thepresence of the inhibitor. In addition, dependence on the αvβ₃ and αvβ₅integrins for adhesion and spreading on VN was demonstrated usingblocking antibodies specific for these integrins. Monoclonal Ab13, whichblocks all β1-containing integrins, was used to show that cellattachment and spreading was not dependent on αvβ₁ integrin. LM609 wasused as a specific block to αvβ₃ and P1F6 was used as a specific blockerof αvβ₅.

siRNA design and transfection. Human specific siRNA against Sdc-1 wastransfected into HAECs at a range of concentrations up to 200 nM. At 4 hafter transfection, each well was supplemented with 3 mL of completegrowth medium; at 24 h post-transfection the cells were lifted intrypsin and expanded in 100 mm tissue-culture plates. Cohorts weredetached with EDTA 24 hr later, resuspended in 100 μL HEPES-buffered DMEsupplemented with 10% FBS and subjected to FACS analysis using mAb B-B4specific for human Sdc-1 and an Alexa-488 conjugated anti-mousesecondary antibody. Cells were analyzed at the University of WisconsinComprehensive Cancer Center Flow Cytometry Facility using a FACSCaliburbenchtop cytometer (BDBiosciences). Cell scatter and propidium iodide(Sigma, 1 μg/sample) staining profiles were used to gate live,single-cell events for data analysis.

Sdc1 and integrin expression in mouse tissues. Frozen sections of normalmouse artery, or tumors arising in the mammary gland of transgenic miceexpression the wnt or β-catenin oncogenes under control of the mammarygland specific MMTV-promoter were fixed and stained with a rabbitpolyclonal antibody specific for Sdc1 and a rat monoclonal antibodyspecific for the endothelial cell marker PECAM. Tissues were alsostained with rabbit polyclonal antibodies to the mouse αv integrinsubunit (AB1930), the mouse β3 integrin subunit (AB1932) or the mouse β5integrin subunit (AB1926). Non-specific control rabbit or rat IgGs wereused to demonstrate specificity. The primary antibodies were stainedwith Alexa488- or Alexa548-conjugated secondary antibodies anddistribution of the receptors in the tissue observed via fluorescencemicroscopy.

Corneal pocket implant angiogenesis assay. Apolyhydroxyethylmethacrylate (PolyHEMA) pellet 1 μl in volume containing100 ng FGF was implanted into pockets surgically prepared in the centerof the avascular corneas of 6 wk-old Balb/c mice. Three mice were usedas controls and three mice received a 0.2 ml Alzet osmotic pumpimplanted subcutaneously on the back of the mice. The pump was loadedwith 100 μM synstatin₈₂₋₁₃₀ in sterile saline and delivers 1 μl into themouse per hr. The mice were allowed to recover and were maintained forseven days. The extent of angiogenesis was recorded by digital cameraimages of the living mouse. Fluorescent dextran was then injected intothe vascular via retro-orbital injection 1-2 minutes prior to sacrifice.Corneas were surgically removed, mounted on slides and the extent ofvascularization observed via fluorescence microscopy.

Example 2 Results

Regulation of αvβ₃ and αvβ₅ integrin activity on endothelial cells byrecombinant syndecan-1 ectodomain. There has not been a concertedexamination of Sdc-1 expression in vascular endothelium. Most reportssuggest that it is expressed poorly or not at all on resting, maturevascular endothelium that line blood vessels. However, there are reportsthat it is expressed on activated endothelial cells participating inangiogenesis in the wounded skin (Elenius et al., 1991; gallo et al.,1996). Sdc-1 is not expressed in endothelial cells lining the rabbitaorta, but expression is upregulated following balloon catheter injuryand persists for up to 12 weeks following injury. There is a report thatSdc-1 is upregulated in a subset of vessels during tumor angiogenesis(Gotte et al., 2002). These studies strongly suggest that Sdc-1 becomesexpressed on activated cells responding to injury or growth factors.Cultured cells, such as human aortic and human umbilical veinendothelial cells reportedly show expression of Sdc-1 at the mRNA level(Mertens et al., 1992), although, as the inventors note above, as theinventors find that expression of the receptor protein in humanumbilical vein endothelial cells is low. However, the expressionpatterns described may be dependent on the growth factors andsupplements, such as brain extract, added to the culture medium.

The inventors have now examined the expression of Sdc1 on several typesof cultured endothelial cells. Flow cytometry or Western blot analysisof cells shows that human aortic endothelial cells (HAECs), human dermalmicrovascular endothelial cells (HMEC-1s), mouse aortic endothelialcells, mouse brain endothelial cells and mouse heart endothelial cellshave a significant cell surface population of Sdc1 (not shown). Inaddition, the inventors examined the expression of Sdc1 in theangiogenic vasculature of mouse tumors. The inventors examined mammarycarcinomas arising in mice that co-express either the wnt1 oncogene orthe β-catenin oncogene. Staining for Sdc1 showed that it is highlyexpressed in the angiogenic tumor vasculature of both tumors, and isco-expressed at this site with the αvβ3 and αvβ5 integrins (FIG. 1).This is in contrast to a normal mouse artery endothelium which showslittle expression of any of these three receptors (FIG. 1).

The inventors have examined the Sdc1 regulation of the αvβ3 and αvβ5integrins in several types of endothelial cells. Human aorticendothelial cells (HAEC) express abundant amounts of the αvβ3 integrinand somewhat less of the αvβ5. The cells also express a high level ofthe β1 integrin subunit, which is capable of assembling with the αvsubunit to form the αvβ1 integrin. All three of these integrins arepotential vitronectin receptors and it is thus not surprising that theseendothelial cells bind and spread on vitronectin. To assess the actualcontribution of each integrin, the inventors have plated the cells onvitronectin in the presence of inhibitory antibodies to the β1 integrins(mAb13), the αvβ3 (LM609) and the αvβ5 (P1F6). The inventors find thatinhibiting the αvβ1 integrin has little or no effect, but that the HAECsfail to attach and spread normally on VN when both the αvβ3 and αvβ5 areinhibited (FIG. 2).

The next test was whether agents that target Sdc1, especiallycompetition with recombinant S1ED, will block the activity of these twointegrins and thus block their interaction with VN. The inventors findthat, indeed, the HAEC cell spreading on VN is blocked by the solubleS1ED at a concentration similar to that of the αvβ3 in mammary carcinoma(Beauvais and Rapraeger, 2003) and that of the αvβ5 integrin in B82Lfibroblasts (McQuade et al., 2006) (FIG. 3). Since the work withinhibitory integrin antibodies demonstrated that both integrins must beblocked on these cells in order to block cell spreading, the inventorsconclude that the recombinant protein must disrupt signaling by both ofthese integrins. The inventors have also performed this analysis withmouse aortic endothelial cells, which express Sdc1, and find that theyare also exquisitely sensitive to competition by the Sdc1 ectodomain,with spreading on VN blocked using 1 μM competitor. The inventors havealso shown this with human microvascular endothelial cells (HMEC-1),which are inhibited in this assay by S1ED with an IC₅₀ of approximately3-10 μM. Secondly, the inventors targeted Sdc1 expression in the cellsusing siRNA. The preliminary experiments demonstrate that at 100 nMsiRNA, which silences Sdc1 expression by 80%, the spreading of the HAECson VN is also disrupted (FIG. 4). Thus, Sdc1 on endothelial cellsappears to be a necessary regulator of the αvβ3 and αvβ5 integrins andthis regulatory activity, and thus the activation of the integrinsthemselves, is blocked by soluble S1ED; this mimics the inhibition thatthe inventors have observed and published with the human mammarycarcinoma cells (Beauvais and Rapraeger, 2003) and B82L fibroblasts(McQuade et al., 2006) and indicates that the protein is a promisinginhibitor of angiogenesis caused by tumors and other human diseases.

Inhibition of angiogenesis in vivo by synstatin. The inventors have nowextended these findings to more direct angiogenesis assays, using asinhibitors the recombinant Sdc1 ectodomain and also using a shorterpeptide encompassing the active site within the Sdc1 ectodomain that theinventors refer to as “synstatin”.

The recombinant Sdc1 ectodomain (S1ED) is comprised of 234 amino acidsextending from the N-terminal signal sequence of the protein to thetransmembrane domain (amino acids 18-252). The recombinant protein isexpressed in bacteria as an epitope-tagged protein (GST fusion proteinor 6×His-tagged protein) for ease of purification on glutathione ornickel columns, respectively.

The peptide that the inventors have developed encompasses amino acids82-130 of the mouse Sdc1 sequence (a 49 amino acid sequence). Thus, thispeptide is referred to as “synstatin₈₂₋₁₃₀”. Mutational analysis of theSdc1 expressed in cells suggests that the active site necessary foractivating the αvβ3 integrin resides in amino acids 88-121 (a 34 aminoacid sequence). The active site for the αvβ5 integrin has not beendefined yet by mutational analysis, but clearly traces to the ectodomainand likely to the same site. Thus, the inventors envisioned thatsynstatin₈₂₋₁₃₀ would contain the active site necessary in Sdc1 forregulating both integrins and that it would compete with this regulatoryactivity on the cells.

The assays that the inventors have used to test the activity ofsynstatin₈₂₋₁₃₀ and to compare it to the native ectodomain (SLED) onwhich its sequence is based are: 1) adhesion of cells to vitronectin,which is a ligand for the αvβ3 and αvβ5 integrins, and 2) growth ofangiogenic blood vessels into the avascular mouse cornea in response tothe implantation of a pellet containing the angiogenic factor FGF—againa process that relies on these integrins.

Inhibition of adhesion and spreading on Vitronectin (VN). The inventorshave tested the adhesion and spreading of MDA-MB-231 mammary carcinomacells on VN in the presence or absence of synstatin₈₂₋₁₃₀. These cellsrely specifically on the active αvβ3 integrin for this adhesion(Beauvais et al., 2004). Recombinant Sdc1 ectodomain (S1ED) has beenshown by the inventors previously to block spreading of these cells withan IC₅₀ of about 3-10 μM (Beauvais et al., 2004). In contrast, theinventors find that synstatin₈₂₋₁₃₀ has an IC₅₀ of about 0.1 μM,indicating that it is about 30-100× more effective (FIG. 5). A 1 μMconcentration of synstatin₈₂₋₁₃₀ completely blocks cell attachment, anoutcome that the inventors have not seen with the highest concentrationsof recombinant S1ED tested (50 μM). As a control, the inventors testedcell adhesion and spreading on fibronectin, a ligand on which the cellsuse the α5β1 integrin and do not rely on the αvβ3 integrin;synstatin₈₂₋₁₃₀ has no effect on attachment and spreading on this ligand(FIG. 5). Thus, synstatin₈₂₋₁₃₀ is a specific and effective inhibitor ofαvβ3 integrin activation.

Similar findings are observed with B82L fibroblasts (FIG. 6). Theinventors have shown previously that these cells rely solely on the αvβ5integrin to recognize VN, and that this recognition also depends onactivation of the integrin by Sdc1 (McQuade et al., 2006). RecombinantSdc1 affects this process with an IC₅₀ of ca. 3-10 μM, similar to itsinhibition of αvβ3 integrin activation in the carcinoma cells (McQuadeet al., 2006). Synstatin₈₂₋₁₃₀ disrupts the spreading of the B82L cellson VN with an IC₅₀ of ca. 0.1 μM, and completely blocks any cellattachment at 1 μM.

Finally, the inventors have examined HMEC-1 cells, which areimmortalized human dermal microvascular endothelial cells. Microvascularendothelial cells are regarded as being the main source of endothelialcells that carry out angiogenesis in vivo. The HMEC-1 cells grown inserum-containing culture medium express Sdc1 (as noted earlier) and alsoexpress active αvβ3 and αvβ5 integrins. The inventors find usinginhibitory antibodies to the αvβ3 and/or αvβ5 integrins that the HMEC-1cells attachment to VN is dependent on these two integrins and both mustbe inhibited to disrupt attachment and spreading on this ligand (notshown), similar to the analysis shown for the HAECs in FIG. 2.Recombinant SLED blocks the spreading on VN with an IC₅₀ of 3-10 μM,similar to its disruption of αvβ3 and/or αvβ5 integrins on the mammarycarcinoma and B82L cells (Beauvais and Rapraeger, 2003; Beauvais et al.,2004; McQuade et al., 2006). Synstatin₈₂₋₁₃₀ is again more effective,disrupting spreading with an IC₅₀ of approximately 0.1 μM (FIG. 6).Thus, the peptide inhibitor synstatin₈₂₋₁₃₀ derived from the nativesequence of Sdc1 is a highly effective inhibitor of these two integrinson endothelial cells as well as other cells that rely on either of thesetwo integrins.

Inhibition of angiogenesis in vivo by disrupting the Sdc1 regulation ofαvβ3 and αvβ5 integrins. To test the efficacy of synstatin₈₂₋₁₃₀ invivo, the inventors have used the mouse corneal pocket implantangiogenesis assay. Here, angiogenesis is induced in the avascular mousecornea by implantation of a polyHEMA pellet containing fibroblast growthfactor (FGF), which is a potent angiogenic agent. The FGF will induceangiogenic outgrowth of new vessels from the limbic vessels at themargin of the cornea into the avascular region at the center of thecornea where the pellet is implanted. The inventors have testedrecombinant S1ED by supplying it along with the FGF in the pelletimplanted into the cornea, and have also introduced it into the systemiccirculation of the mouse through the use of an osmotic pump placedsubcutaneously on the back of the animal. The latter protocol moreclosely mimics the manner in which such an inhibitor might be deliveredas a drug in patients. In both circumstances recombinant S1ED blocks theFGF-induced angiogenesis. The concentration necessary for the block invivo has not yet been measured. It is supplied at a concentration of 2.4mM in the osmotic pump, which delivers 1 μl into the mouse per hour; ifthe inventors assume that it is contained within the 2 ml blood volumeof the mouse, and assume that there is no protein degradation orclearance, then it would achieve concentrations of approximately 29 μMafter 24 hr. However, this is likely to be a high estimate, as clearanceand degradation are likely to occur, and the protein is likely to bepresent in the greater volume of the mouse than just the vascularsystem. Thus, the recombinant protein may be highly effective in vivo atconcentrations of 29 μM or less.

The inventors have tested synstatin₈₂₋₁₃₀ in the corneal angiogenesisprotocol as well, delivering it into the mouse circulation via theosmotic pump as described for S1ED. The starting concentration in thepump is 100 μM, 24-fold lower than S1ED; using the same assumptions andcaveats as noted for pump delivery of recombinant S1ED, the systemicconcentration of the inhibitor may reach 1.2 μM after 24 hr. Again, thisis likely a high estimate. In six mice tested to date, the inventorshave seen a complete inhibition of angiogenesis via systemic delivery ofsynstatin₈₂₋₁₃₀. Control mice containing the FGF implant show extensivevessel outgrowth extending from the limbus vessel at the perimeter ofthe cornea to the pellet in the center, where the new angiogenic vesselsengulf the FGF pellet (FIG. 7). In mice with systemically-deliveredsynstatin₈₂₋₁₃₀, the mice show no angiogenesis (FIG. 7). The inventorshave not yet tried lower concentrations. It should be emphasized thatfollowing the one week of treatment with this concentration of inhibitorthat is highly effective against angiogenesis, these mice show no illeffects. They are active, eat normally, maintain weight and show noaltered behavior.

Example 3

Introduction. Angiogenesis, or the sprouting of new blood vessels fromexisting ones, occurs in development and in diseases such as diabeticretinopathy, endometriosis, and tumor-induced angiogenesis. The mature,resting endothelial cells in the donor vessels are activated to progressthrough an angiogenic program, in which they undergo proliferation andinvasion, maturation, and apoptosis; the latter, also known as “vascularpruning” is especially important in molding the architecture of the newvessels (Bergers and Benjamin, 2003; Stupack and Cheresh, 2003).

FGF and VEGF, two growth factors often released by tumors, are potentangiogenic factors. Their activities are closely tied to the activity oftwo integrins, the α_(v)β₃ and α_(v)β₅ integrins (Stupack and Cheresh,2003), which have roles in the chemotactic migration and in the survivalof the endothelial cells. The expression of these two integrins isinduced by FGF and VEGF signaling and the integrins and growth factorreceptors then collaborate in the signaling pathways leading toangiogenesis. Uncoupling of this signaling by inactivation of eithertype of receptor leads to apoptosis of the endothelial cells (Stupack etal., 2001) and this mechanism is believed to have a major role in vesselpruning as the new vasculature acquires its final architecture. Theα_(v)β₃ and α_(v)β₅ integrins are not generally expressed in adultcells, with the exception of a few sites such as osteoclasts, but theirexpression on activated endothelial cells during angiogenesis (Byzova etal., 1998) as well as in many tumors during metastasis, makes themattractive targets for combating tumorigenesis. A choice target is theirapoptotic role, as triggering apoptosis of the endothelial cells willstarve the tumors that require a new vascular supply for nutrient andgaseous exchange. Indeed, this apoptotic role of the integrin mayexplain why β₃ and β₅ knockout mice exhibit increased angiogenesis,whereas inhibitors of the α_(v)β₃ and α_(v)β₅ integrins in normal miceare anti-angiogenic; it is hypothesized that potent apoptotic signalingarises from these integrins when they are inhibited in wild-type mice,whereas the integrin null mice are freed from this apoptotic mechanism(Bader et al., 1998; Reynolds et al., 2002).

The activation of an integrin typically refers to a conformation changethat allows ligand binding. The α_(v)β₃ has been used as a model tounderstand this activation mechanism. It proceeds through at least twoactivation states, each resulting in a change in conformation of theextracellular domain in response to intracellular signals andextracellular ligand binding (Boettiger et al., 2001; Boettiger et al.,2001; Du et al., 1991; Frelinger et al., 1991; Humphries, 1996;Liddington and Ginsberg, 2002; Pelletier et al., 1996; Plow et al.,2000; Xiong et al., 2001; Yan et al., 2000). “Activating” or“inactivating” antibodies directed to the integrin extracellular domainserve to confine it to one or the other conformation. One such antibody,LM609, freezes the α_(v)β₃ integrin in the inactive conformation, blocksangiogenesis and leads to endothelial cell apoptosis. A humanizedversion of this antibody (Vitaxin) is in clinical trials as an antitumoragent.

The syndecans are multifunctional matrix receptors on the surface of alladherent cells. They anchor to the matrix via heparan sulfateglycosaminoglycan chains and communicate to the cytoplasm via short buthighly conserved cytoplasmic domains. The heparan sulfate chains haveimportant signaling properties, as they enhance the assembly of growthfactor with their receptor tyrosine kinases. However, it is becomingclear that the syndecan proteins have important regulatory roles aswell, often leading to the description of this family as “co-receptors,”as they assemble with and control the signaling of other receptors onthe cell surface. Several reports now indicate that specialized sitesmay exist within the syndecan extracellular protein domains, which ifmutated or targeted with antibodies, disrupt tumor cell invasion. Iftrue, such sites may hold promise as targets for therapeutic drugs tocombat tumorigenesis.

Syndecan-1 associates with the α_(v)β₃ and α_(v)β₅ integrins, and thisassociation is disrupted by SSTN. The inventors have described a rolefor syndecan-1 in regulating activation of the α_(v)β₃ integrin in humanmammary carcinoma cells, and the α_(v)β₅ integrin in B82L mousefibroblasts. This regulation involves a site in the extracellular domainof syndecan-1, demonstrated in human MDA-MB-231 or MB-435 mammarycarcinoma cells, which utilize the α_(v)β₃ integrin to bind, spread ormigrate on vitronectin. Integrin activity is abolished by silencingsyndecan-1 expression with human-specific siRNA, and is rescued byre-expressing mouse syndecan-1; however, deletion mutants lacking aregion of the extracellular domain encompassing amino acids 88-121 inthe mouse sequence fail to rescue (FIG. 8A). This sequence has a highdegree of conservation across mouse, hamster, rat and human syndecan-1(FIG. 9).

Because syndecan-1 regulates both of these integrins that are attractivetargets for anti-angiogenic therapy, the inventors questioned whetherthis regulatory mechanism existed on endothelial cells. Although somereports indicate that syndecan-1 is not expressed on vascularendothelium (Elenius et al., 1991; Gallo et al., 1996; Hayashi et al.,1987; Kainulainen et al., 1996), other reports suggest that it isupregulated on activated endothelial cells undergoing angiogenesis(Elenius et al., 1991; Gallo et al., 1996; Gotte et al., 2002;Kainulainen et al., 1996). The inventors examined the expression ofsyndecan-1 and the two integrins on three lines of vascular endothelialcells by flow cytometry (FIG. 8B). Human aortic endothelial cells andhuman dermal microvascular endothelial cells all express modest levelsof syndecan-1 and a larger population of the α_(v)β₃ and α_(v)β₅integrins. Mouse aortic endothelial cells express higher levels ofsyndecan-1 than their human counterparts, but less of the α_(v)β₃integrin. Although a reliable antibody for detecting the mouse α_(v)β₅integrin by flow is not available, it can be shown that they alsoexpress the α_(v)β₅ integrin by western blot, shown in comparison toB82L fibroblasts (FIG. 8B), known to express this integrin.

To determine if the syndecan and integrins are in a regulatory complex,blots containing syndecan-1 immunoprecipitated from HMECs were probedfor the co-precipitation of the β₃ and β₅ integrin subunits; this isindicative of an association of the syndecan with the α_(v)β₃ andα_(v)β₅ integrins as these β subunits associate only with the α_(v)subunit at endothelial cell surfaces. Both integrins co-precipitate withthe syndecan and in seemingly equal proportions suggestive of a roughly1:1 correspondence (FIG. 8C). Next, to determine if the interaction isdependent on the site identified in the syndecan-1 ectodomain, the cellswere preincubated with either GST-mSED, a recombinant GST fusion proteincontaining the ectodomain of mouse syndecan-1, or a peptide (called“synstatin” or SSTN) containing the active site in syndecan-1. Mousesyndecan-1 ectodomain is used as it is not recognized by thehuman-specific monoclonal antibodies used to immunoprecipitate thesyndecan. Both the recombinant protein and the SSTN peptide compete withthe interaction and displace the integrins from the human syndecan-1expressed on the endothelial cells (FIG. 8C). Lastly,immunoprecipitations were performed using MDA-MB-231 human mammarycarcinoma cells expressing either native mouse syndecan-1 in addition tothe endogenous human counterpart, or a mouse mutant containing a Δ67-121deletion that removes the putative active site at 88-121. The α_(v)β₃integrin expressed by these cells co-immunoprecipitates with either thehuman or the mouse syndecan-1, these interactions are competed by eitherthe full-length recombinant S1ED or the SSTN peptide, and the integrinfails to associate with the mouse mutant lacking the active site (FIG.8D). Thus, syndecan-1 appears to associate with the α_(v)β₃ and α_(v)β₅integrin in vascular endothelial cells as a putative regulatorymechanism that can be disrupted by SSTN.

The α_(v)β₃ and α_(v)β₅ integrin on vascular endothelial cells areregulated by syndecan-1. The inventors next questioned whether SSTN isinhibitory to integrin activation. HMECs plated on a substratumconsisting solely of a monoclonal antibody (B-B4) to syndecan-1 attachto the antibody and spread. Their prior work has suggested that the cellspreading requires integrin activation and signaling. Indeed, theα_(v)β₃ integrin on the cells is activated, detected by staining thecells with the ligand-mimetic antibody WOW-1 (FIG. 10A); this antibodybinds only to activated integrin. However, co-incubation of the cellswith 0.5 μM SSTN prevents both the cell spreading and recognition byWOW-1, indicating that competitive displacement of the integrin from theligated syndecan prevents integrin activation. Identical results areseen in the converse experiment, namely plating the cells onvitronectin, a ligand for both the α_(v)β₃ and α_(v)β₅ integrins. Thespreading of the cells is dependent on both integrins; spreading isblocked in the presence of antibodies LM609 and P1F6 (FIG. 10A), whichinactive the α_(v)β₃ and α_(v)β₅ integrins, respectively, but is notblocked in the presence on either antibody alone (not shown). However,addition of either 5.0 μM recombinant mS1ED or 0.5 μM SSTN blocksspreading as effectively as the combined antibody treatment (FIG. 10A).An identical result is obtained if syndecan-1 expression is silencedwith siRNA (FIG. 10B). Both findings support the conclusion thatsyndecan-1 is simultaneously regulating the activation of both integrinson vascular endothelial cells.

The effective inhibitory concentration of SSTN is lower than that of therecombinant mS1ED protein. This is shown using either HMECs or humanaortic endothelial cells plated on vitronectin and quantifying cellspreading (FIG. 10C) or cell attachment (FIG. 10D). Competition withmS1ED at 1-3 μM concentrations reduced cell spreading by over 80-90%,respectively for the HMECs, and displays at IC50 of about 1 μM forHAECs, which are more resistant than the HMECs. Complete integrininactivation necessary for blocking cell attachment altogether requiresover a 10-fold higher concentration of 10 μM. In comparison, SSTNdisplays inhibitory activity equal to that of mS1ED when used at a10-fold lower concentration and has significant inhibitory properties inthe 0.1-0.3 μM range. At this concentration it is equal or moreeffective than 10 μg/ml of the inhibitory antibodies LM609 and PIF6.Although the reason for the greater inhibition by SSTN is not known, itmay trace to misfolding of the mS1ED protein when expressed in bacteria,or it may indicate that the SSTN sequence in the full ectodomain ispartially hidden. Computer modeling of the syndecan or SSTN sequence isnot reliable as the proteins have little or no homology to any proteinsthat have been crystallized to date.

Syndecan-1 is expressed during angiogenesis. To test the activity ofSSTN as an anti-angiogenic agent, the inventors turned to the in vitroaortic ring outgrowth assay and the in vivo corneal angiogenesis model.The aortic outgrowth assay allows quantification of microvesseloutgrowth from segments of mouse aorta explanted to type I collagengels. As was found when examining the mouse aortic endothelial cell linein culture, both syndecan-1 and the α_(v)β₃ and α_(v)β₅ integrins appearpresent even on the resting mouse aorta (FIG. 11A). In addition,microvessels growing out from the aortic explant over a 7 days period inresponse to FGF stain positively for syndecan-1, with appearsco-expressed with the α_(v)β₃ and α_(v)β₅ integrins (FIG. 11B). Toextend this finding of syndecan-1 expression in activated endothelium,the inventors also examined two models of mouse breast tumor formation.Tumors derived from wnt-1 overexpression in the mammary gland show ahigh degree of vascularization, identified as internal networks lined bya thick cell layer than stains intensely for syndecan-1 and for PECAM(CD31), indicative of activated endothelium (FIG. 11C). In fact,syndecan-1 expression in these cells swamps out the positive stainingfor syndecan-1 seen in the mammary epithelial cells, which are known tobe positive for syndecan-1. These same cell layers as positive forα_(v), β₃ and β₅ integrin subunits. A similar finding is seen in tumorsarising from overexpression of β-catenin (FIG. 11C). Although thevascularization is not as extensive as in the β-catenin-induced tumors,there are clear thickened layers of cells that are PECAM, syndecan-1 andα_(v)β₃/α_(v)β₅ integrin positive in these mammary tumors.

SSTN inhibits angiogenesis in vitro and in vivo. To test the effect ofSSTN on microvessel outgrowth, segments of thoracic aorta were explantedto collagen gels in the presence of either 50 ng/ml vascular endothelialcell growth factor (VEGF) or 30 ng/ml FGF-2, and incubated in thepresence of either recombinant mS1ED or SSTN. Although some vesseloutgrowth is observed in the absence of exogenous angiogenic agents,likely due to VEGF release by supporting cells growing out from theexplant, the outgrowth is greatly (≧10-fold) increased by FGF or VEGF(FIG. 12). Addition of recombinant mS1ED blocks VEGF-induced outgrowthby more than 70% at 10 μM, and greater than 95% at 30 μM. FGF inducedoutgrowth requires slightly higher inhibitory concentrations(approximately 30 μM mS1ED for 70% inhibition), which may trace to adifference in susceptibility of the two integrins, as VEGF signaling isreportedly coupled to α_(v)β₅ integrin activation and FGF signaling iscoupled to α_(v)β₃ activation. SSTN is 10-fold more potent as ananti-angiogenic agent, displaying an IC50 of 0.1 μM in FGF and aslightly greater IC50 in response to VEGF. These concentrations are muchin line with the IC50's displayed in the in vitro cell attachment andspreading assays with HMECs and HAECs. It should be noted that themicrovessel outgrowth is accompanied by the outgrowth of support cells,most likely smooth muscle cells and fibroblasts. These cells are notendothelial cells, as they do not stain positively for PECAM, and theiroutgrowth is unaffected by mS1ED or SSTN (FIG. 12). Thus, the effects ofSSTN are highly localized to the vascular endothelial cells.

Lastly, SSTN was tested in the in vivo corneal angiogenesis assay.Polyhydroxyethylmethylacrylate pellets (0.25 μL) containing 67 ng FGF-2and sucralfate as a slow release agent were implanted into the avascularmouse cornea. After seven days, fluorescent dextran was injectedsuborbitally to highlight the vascular system and the mice weresacrificed. Visual inspective of either the eye or the dissected corneashows significant vessel ingrowth towards the implanted pellet from thelimbic vessel at the margin of the cornea. Initial experiments foundthat incorporation of recombinant mS1ED into the pellet reduced theangiogenesis (data not shown). To supply the inhibitor in a morequantifiable and physiological manner, recombinant mS1ED or SSTN weredelivered systemically via Alzet osmotic pumps implanted subcutaneouslyon the backs of the animals. Pumps containing either 2500 μM recombinantmS1ED or 30 μM SSTN achieved a nearly total block of angiogenesis.Testing a range of SSTN concentration in the pump showed that 50%inhibition of angiogenesis is achieved by 3-10 μM SSTN concentrations inthe pump (FIG. 13). To correlate this with the active concentration inthe plasma, the inventors took advantage of rabbit polyclonal antibodiesgenerated against mS1ED, which detect active SSTN on dot blots. Theantibodies appear to recognize an active conformation of SSTN, asheating the peptide to 95° C. causes it to lose its inhibitory activityand its recognition by antibodies on dot blots (data not shown). Usingthis method to detect active SSTN in the blood collected from animalsafter 1 week of SSTN treatment, SSTN is shown to be in the blood at125-150 nM when present at 10 μM in the osmotic pump. This correlateswell with the IC50 of ca. 0.1 μM observed in vitro and suggests aclearance rate of 10-13 hr in the plasma.

Effective size of SSTN peptide. The SSTN peptide that we have used isSSTN₈₂₋₁₃₀, based on the mouse sequence (cf. FIG. 9). This peptide spansthe active site in mouse syndecan-1 and retains flanking amino acids oneither side in case they are required to maintain structure. The peptidecontains two adjacent regions in which the amino acids are highlyconserved across species, suggestive of a conserved function (FIG. 14).Truncation of SSTN₈₂₋₁₃₀ to smaller peptides that delete portions ofthese conserved sites cause loss of activity (FIG. 14). Thus,SSTN₈₈₋₁₂₁, which retains on amino acid C-terminal and one amino acidN-terminal to the conserved domain is active, but SSTN₈₈₋₁₁₇, whichremoves four of the conserved amino acids at the C-terminus is 3-foldless active in cell attachment and spreading assays on VN. Thus, itappears that the most active SSTN peptide is that which retains both ofthese conserved sequences.

Effects of SSTN on CAG myeloma tumor formation in vivo. The inventorshave introduced human CAG myeloma cells expressing luciferase intoimmunodeficient SCID mice and treated the mice either with SSTN orcontrol PBS. 10⁵ cells were injected subcutaneously into the haunches of10 animals and allowed to grow for 10 days. At this point, tumors havebegun to form and can be detected by manual palpation or by imaging theluciferase-expressing tumor cells. Alzet pumps containing either PBS or100 μM SSTN were implanted on the backs of the animals and the tumorsallowed to grow for an additional 28 days. The pumps are 28-day pumps(unlike the 7-day pumps used for corneal angiogenesis assays) anddelivered 0.25 μL of 100 μM SSTN per hr. This is roughly equivalent tothe corneal angiogenesis assays in which the 7 day pumps deliver 1 μL of30 μM SSTN per hr, which achieves significant inhibition ofangiogenesis. After the 28 day treatment period, the tumors were againimaged in situ, indicating that the 5 control tumors were 10-11 timeslarger than tumors in the SSTN-treated animals (FIG. 15). The tumorswere dissected and weighed, again showing that the SSTN reduced by tumorsize by over 10-fold (FIG. 16). In addition, the SSTN-treated tumorsappear pale, suggesting a lack of vascularization. Sectioning of thetumors and staining for mouse CD34, an endothelial cell marker, tomonitor the ingrowth of host blood vessels into the human tumor shows asignificant reduction in vessel density and vessel length. There is nopositive staining using antibody specific for human CD34, indicatingthat the tumors are vascularized by host angiogenesis and that this isblocked by circulating SSTN.

Summary. These results show that SSTN is an effective inhibitor of theα_(v)β₃ and α_(v)β₃ integrins both in vitro and in vivo. Syndecan-1 isexpressed together with these integrins on activated endothelial cellsundergoing angiogenesis in response to FGF and VEGF, or in tumors. Theintegrins appear to rely on syndecan-1 to undergo activation, asblocking syndecan-1 expression, deleting the integrin activation sitefrom the syndecan receptor, or, as shown here, competing with theinteraction of the syndecan with the integrins using a peptide (SSTN)containing the syndecan active site, all serve to inactivate theintegrins and block endothelial cell attachment, spreading, andangiogenesis. SSTN (or SSTN mimetics) is an attractive therapeutic totarget and inactivate these two integrins on tumor cells, activatedendothelial cells, osteoclasts, and other cells that depend on theseintegrins in disease processes.

Example 4

The synstatin (SSTN) peptides that were previously tested have beenderived from the mouse syndecan-1 (Sdc1) sequence so that theirbiological activity can be tested in mouse models without eliciting animmune response. Nonetheless, the in vitro work with cell lines showsthat both the mouse and human Sdc1 have identical abilities to regulatethe αvβ3 and αvβ5 integrins.

To test a human SSTN peptide, experiments were conducted with human SSTNwith amino acids 88-121 of the human sequence (hSSTN 88-121) (SEQ IDNO:28) and amino acids 89-120 of the human sequence (hSSTN 89-120) (SEQID NO:21). The human and mouse Sdc1 sequences differ in length by oneamino acid counting from the amino terminus to the beginning of the SSTNsequence (human is longer by one amino acid), and therefore, it wasdesirable to test not only the 88-121 sequence, but also 89-120 to makesure that the difference of one amino acid on either end would notaffect its activity.

In FIG. 17, both peptides show identical abilities to block theattachment and spreading of human MDA-MB-231 breast carcinoma cells tovitronectin (VN). The inventors have shown previously that thisattachment and spreading is dependent on Sdc1 activating the α_(v)β₃integrin on these cells (Beauvais et al., 2004). Furthermore, the twopeptides show an IC50 of 0.1 to 0.3 μM, which is identical to the mostactive mouse SSTN peptides (mouse SSTN 82-130 or mouse SSTN 88-122.) Thepeptides have no effect on cell attachment and spreading on fibronectin(FN) when used at a concentration 100-fold greater than their IC50. Theinventors have shown previously (Beauvais et al., 2004) that attachmentand spreading on FN relies on the α₅β₁ integrin and is not dependent onSdc1.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 4,196,265-   U.S. Pat. No. 4,554,101-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 5,302,523-   U.S. Pat. No. 5,322,783-   U.S. Pat. No. 5,384,253-   U.S. Pat. No. 5,399,363-   U.S. Pat. No. 5,440,013-   U.S. Pat. No. 5,446,128-   U.S. Pat. No. 5,464,765-   U.S. Pat. No. 5,466,468-   U.S. Pat. No. 5,475,085-   U.S. Pat. No. 5,538,877-   U.S. Pat. No. 5,538,880-   U.S. Pat. No. 5,543,158-   U.S. Pat. No. 5,550,318-   U.S. Pat. No. 5,563,055-   U.S. Pat. No. 5,580,859-   U.S. Pat. No. 5,589,466-   U.S. Pat. No. 5,610,042-   U.S. Pat. No. 5,618,914-   U.S. Pat. No. 5,641,515-   U.S. Pat. No. 5,656,610-   U.S. Pat. No. 5,670,155-   U.S. Pat. No. 5,672,681-   U.S. Pat. No. 5,674,976-   U.S. Pat. No. 5,702,932-   U.S. Pat. No. 5,710,245-   U.S. Pat. No. 5,736,524-   U.S. Pat. No. 5,780,448-   U.S. Pat. No. 5,789,215-   U.S. Pat. No. 5,840,833-   U.S. Pat. No. 5,846,225-   U.S. Pat. No. 5,846,233-   U.S. Pat. No. 5,859,184-   U.S. Pat. No. 5,928,906-   U.S. Pat. No. 5,929,237-   U.S. Pat. No. 5,945,100-   U.S. Pat. No. 5,981,274-   U.S. Pat. No. 5,994,624-   Adams et al., J. Cell Biol., 152:1169-1182, 2001.-   Akiyama et al., J. Cell Biol., 109:863-875, 1989.-   Albert et al., Nat. Cell Biol., 2:899-905, 2000.-   Alexander et al., Nat. Genet., 25:329-332, 2000.-   Anttonen et al., Br. J. Cancer, 79:558-564, 1999.-   Ausubel et al., In: Current Protocols in Molecular Biology, John,    Wiley & Sons, Inc, New York, 1994.-   Baciu and Goetinck, Mol. Biol. Cell, 6:1503-1513, 1995.-   Bader et al., Cell, 95:507-519, 1998.-   Baichwal and Sugden, In: Gene Transfer, Kucherlapati (Ed.), New    York, Plenum Press, 117-148, 1986.-   Barany and Merrifield, In: The Peptides, Gross and Meienhofer    (Eds.), Academic Press, New York, pp. 1-284 1979.-   Barbareschi et al., Cancer, 98:474-483, 2003.-   Bayer-Garner et al., J. Cutan. Pathol., 28:135-139, 2001.-   Beauvais and Rapraeger, Exp. Cell Res., 286:219-232, 2003.-   Beauvais and Rapraeger, Exp. Cell Res., 286:219-232, 2004.-   Beauvais and Rapraeger, Reprod. Biol. Endocrinol., 2:3, 2004.-   Beauvais et al., J. Cell Biol., 167(1):171-81, 2004.-   Bergers and Benjamin, Nature Rev. Cancer, 3:401-410, 2003.-   Bernfield et al., Annu. Rev. Biochem., 68:729-777, 1999.-   Boettiger et al., J. Biol. Chem., 276:31684-31690, 2001.-   Boettiger et al., Molec. Biol. Cell, 12:1227-1237, 2001.-   Boudreau et al., J. Cell Biol., 139(1):257-264, 1997.-   Brooks et al., Cell, 79:1157-1164, 1994.-   Brooks et al., J. Clin. Invest., 99:1390-1398, 1997.-   Brooks et al., Science, 264:569-571, 1994.-   Burbach et al., Matrix Biol., 22:163-177, 2003.-   Byzova et al., Thromb. Haemost., 80:726-734, 1998.-   Capaldi et al., Biochem. Biophys. Res. Comm., 74(2):425-433, 1977.-   Carey et al., Exp. Cell Res., 214:12-21, 1994.-   Carey et al., J. Cell Biol., 124:161-170, 1994.-   Carman and Springer, Curr. Opin. Cell Biol., 15:547-556, 2003.-   Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987.-   Colorado et al., Cancer Res., 60(9):2520-2526, 2000.-   Conejo et al., Int. J. Cancer, 88:12-20, 2000.-   Couchman et al., Int. Rev. Cytol., 207:113-150, 2001.-   Coupar et al., Gene, 68:1-10, 1988.-   Crescimanno et al., J. Pathol., 189:600-608, 1999.-   De et al., J. Biol. Chem., 278:39044-39050, 2003.-   Degryse et al., Oncogene, 20:2032-2043, 2001.-   Du et al., Cell, 65:409-416, 1991.-   Elenius et al., J. Cell Biol., 114(3): 585-595, 1991.-   Elenius et al., J. Cell Biol., 114:585-595, 1991.-   Eliceiri and Cheresh, J. Clin. Invest., 103:1227-1230, 1999.-   Eliceiri et al., J. Cell Biol., 140:1255-1263, 1998.-   Eliceiri, Circ. Res., 89:1104-1110, 2001.-   Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987.-   Felding-Habermann and Cheresh, Curr. Opin. Cell. Biol., 5:864-868,    1993.-   Felding-Habermann et al., Proc. Natl. Acad. Sci. USA, 98:1853-1858,    2001.-   Finnemann, Adv. Exp. Med. Biol., 533:337-342, 2003b.-   Finnemann, Embo. J., 22:4143-4154, 2003a.-   Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352, 1979.-   Frelinger et al., J. Biol. Chem., 266:17106-17111, 1991.-   Friedlander et al., Science, 270:1500-1502, 1995.-   Friedmann, Science, 244:1275-1281, 1989.-   Fujiya et al., Jpn. J. Cancer Res., 92:1074-1081, 2001.-   Gallo et al., J. Invest. Derm., 107:676-683, 1996.-   Gallo et al., J. Invest. Dermatol., 107(5):676-683, 1996.-   Gao et al., J. Cell Biol., 135:533-544, 1996.-   Giancotti and Ruoslahti, Science, 285:1028-1032, 1999.-   Gopal, Mol. Cell Biol., 5:1188-1190, 1985.-   Gotte et al., Invest. Ophthal. Visual Sci., 43(4):1135-1141, 2002.-   Gotte et al., IOVS, 43:1135-1141, 2002.-   Graham and Van Der Eb, Virology, 52:456-467, 1973.-   Granes et al., Exp. Cell Res., 248:439-456, 1999.-   Hall et al., Exp. Eye Res., 77:281-286, 2003.-   Hansen et al., J. Cell Biol., 126:811-819, 1994.-   Harland and Weintraub, J. Cell Biol., 101:1094-1099, 1985.-   Harlow and Lane, In: Antibodies, A Laboratory manual, Cold Spring    Harbor Laboratory, 1988.-   Hayashi et al., J. Histochem. Cytochem., 35:1079-1088, 1987.-   Hermonat and Muzycska, Proc. Natl. Acad. Sci. USA, 81:6466-6470,    1984.-   Hirabayashi et al., Tumour Biol., 19:454-463, 1998.-   Hood et al., J. Cell Biol., 162:933-943, 2003.-   Horwich et al., Virol., 64:642-650, 1990.-   Humphries, Curr. Opin. Cell Biol., 8:632-640, 1996.-   Iba et al., J. Cell Biol., 149:1143-1156, 2000.-   Ilic et al., J. Cell Biol., 143:547-560, 1998.-   Inki and Jalkanen, Ann. Med., 28:63-67, 1996.-   Inki et al., Br. J. Cancer, 70:319-323, 1994.-   Inki et al., J. Pathol., 172:349-355, 1994.-   Izzard et al., Exp. Cell Res., 165:320-336, 1986.-   Johannesson et al., J. Med. Chem., 42(22):4524-4537, 1999.-   Johnson et al., In: Biotech. Pharm., Pezzuto et al. (Eds.), Chapman    and Hall, NY, 1993.-   Jones et al., J. Oral. Pathol. Med., 26:63-68, 1997.-   Kaeppler et al., Plant Cell Reports 9: 415-418, 1990.-   Kainulainen et al., J. Biol. Chem., 271:18759-18766, 1996.-   Kamphaus et al., J. Biol. Chem., 275(2):1209-1215, 2000.-   Kamphaus et al., J. Biol. Chem., 278:1209-1215, 2000.-   Kaneda et al., Science, 243:375-378, 1989.-   Kato et al, J. Biol. Chem., 266:3361-3364, 1991.-   Kato et al., Mol. Biol. Cell, 6:559-576, 1995.-   Khan et al., J. Biol. Chem., 263:11314-113148, 1988.-   Kim et al., Mol. Biol. Cell, 5:797-805, 1994.-   Kim et al., Oncogene, 22:826-830, 2003.-   Klass et al., J. Cell Sci., 113:493-506, 2000.-   Klatka, Eur. Arch. Otorhinolaryngol., 259:115-118, 2002.-   Kumar-Singh et al., J. Pathol., 186:300-305, 1998.-   Kyte and Doolittle, J Mol Biol, 157(1):105-32, 1982.-   Lebakken and Rapraeger, J. Cell Biol., 132:1209-1231, 1996.-   Lebakken, and Rapraeger, J. Cell Biol., 132:1209-1221, 1996.-   Leppa et al. Cell Regul., 2:1-11, 1991.-   Leppa et al., J. Cell Sci., 109:1393-1403, 1996.-   Leppa et al., Proc. Natl. Acad. Sci. USA, 89:932-936, 1992.-   Levy et al., Br. J. Cancer, 74:423-431, 1996.-   Levy et al., Bull. Cancer, 84:235-237, 1997.-   Liapis et al., Diagn. Mol. Pathol., 5:127-135, 1996.-   Liddington and Ginsberg, J. Cell Biol., 158:833-839, 2002.-   Lindberg et al., J. Cell Biol., 134:1313-1322, 1996.-   Liu et al., J. Biol. Chem., 273:22825-22832, 1998.-   Maeshima et al., J. Biol. Chem., 275(28):21340-21348, 2000.-   Maniatis, et al., In: Molecular Cloning, A Laboratory Manual, Cold    Spring Harbor Press, NY, 1990.-   Matsumoto et al., Int. J. Cancer, 74:482-491, 1997.-   McFall and Rapraeger, J. Biol. Chem., 272:12901-12904, 1997.-   McFall and Rapraeger, J. Biol. Chem., 273:28270-28276, 1998.-   McFall and Rapraeger, J. Biol. Chem., 273:28270-28276, 1998.-   McLean et al., J. Biol. Chem., 265:17126-17131, 1990.-   McQuade and Rapraeger, J. Biol. Chem., 278:46607-46615, 2003.-   McQuade et al., J. Cell Sci., Syndecan-1 regulates {alpha}v{beta}5    integrin activity in B82L fibroblasts, May, 2006 [Epub ahead of    print].-   Memmo and McKeown-Longo, J. Cell Sci., 111(Pt 4):425-433, 1998.-   Merrifield, Science, 232: 341-347, 1986.-   Mertens et al., J. Biol. Chem., 267(28):20435-20443, 1992.-   Miranti and Brugge, Nat. Cell Biol., 4:E83-90, 2002.-   Mundhenke et al., Am. J. Pathol., 160:185-194, 2002.-   Myers et al., Am. J. Pathology, 161(6): 2099-2109, 2002.-   Myers et al., J. Cell Biol., 148(2): 343-351, 2000.-   Nakaerts et al., Int. J. Cancer, 74:335-345, 1997.-   Nakanishi et al., Intl. J. Cancer, 80:527-532, 1999.-   Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982.-   Nicolau et al., Methods Enzymol., 149:157-176, 1987.-   Numa et al., Int. J. Oncol., 20:39-43, 2002.-   Oh et al., J. Biol. Chem., 272:11805-11811, 1997b.-   Oh et al., J. Biol. Chem., 272:8133-8136, 1997a.-   Oh et al., J. Biol. Chem., 273:10624-10629, 1998.-   Ohtake et al., Br. J. Cancer, 81:393-403, 1999.-   O'Reilly et al., Cell, 79(2):315-328, 1994.-   O'Reilly et al., Cell, 88(2):277-285, 1997.-   Panetti et al., J. Biol. Chem., 270:18593-18597, 1995.-   Park et al., J. Biol. Chem., 277:29730-29736, 2002.-   Pasqualini et al., J. Cell Sci., 105(Pt 1):101-11, 1993.-   PCT Appln. WO 94/09699-   PCT Appln. WO 95/06128-   Pelletier et al., J. Biol. Chem., 271:1364-1370, 1996.-   Petitclerc et al., Cancer Res., 59:2724-2730, 1999.-   Pilch et al., J. Biol. Chem., 277:21930-21938, 2002.-   Plow et al., J. Biol. Chem., 275:21785-21788, 2000.-   Plow et al., J. Biol. Chem., 275:21785-21788, 2000.-   Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985.-   Potter et al., Proc. Natl. Acad. Sci. USA, 81:7161-7165, 1984.-   Pulkkinen et al., Acta Otolaryngol., 117:312-315, 1997.-   Rapraeger and Ott, Curr. Opin. Cell Biol., 10:620-628, 1998.-   Rapraeger et al., J. Cell Biol., 103:2683-2696, 1986.-   Rapraeger, J. Cell Biol., 149:995-998, 2000.-   Ratnikov et al., J. Biol. Chem., 277:7377-7385, 2002.-   Remington's Pharmaceutical Sciences, 15^(th) ed., pages 1035-1038    and 1570-1580, Mack Publishing Company, Easton, Pa., 1980.-   Reynolds et al., Nature Med., 8:27-34, 2002.-   Reynolds et al., Nature Med., 8:27-34, 2002.-   Ridgeway, In: Vectors: A survey of molecular cloning vectors and    their uses. Rodriguez and Denhardt (Eds.), Stoneham, Butterworth,    467-492, 1988.-   Rintala et al., Gynecol. Onol., 75:372-378, 1999.-   Rippe et al., Mol. Cell Biol., 10:689-695, 1990.-   Roskelley et al., Curr. Opin. Cell Biol., 7:736-747, 1995.-   Sambrook et al., In: Molecular Cloning, A Laboratory Manual, 3rd    Ed., Cold Spring Harbor Press, NY, 2001.-   Sambrook et al., In: Molecular Cloning, A Laboratory Manual, 3rd    Ed., Cold Spring Harbor Press, NY, 2000.-   Sanderson and Bernfield, Proc. Natl. Acad. Sci. USA, 85:9562-9566,    1988.-   Sanderson and Borset, Ann. Hematol., 81:125-135, 2002.-   Sanderson, Semin. Cell Dev. Biol., 12:89-98, 2001.-   Saoncella et al., Proc. Natl. Acad. Sci. USA, 96:2805-2810, 1999.-   Singer et al., J. Cell Biol., 104:573-584, 1987.-   Soldi et al., Embo J., 18:882-892, 1999.-   Soukka et al., J. Oral Pathol. Med., 29:308-313, 2000.-   Stanley et al., Am. J. Clin. Pathol., 112:377-383, 1999.-   Stewart and Young, In: Solid Phase Peptide Synthesis, 2d. Ed.,    Pierce Chemical Co. 1984.-   Streeter and Rees, J. Cell Biol., 105:507-515, 1987.-   Stupack and Cheresh, Oncogene, 22:9022-9029, 2003.-   Stupack et al., J. Cell Biol., 155:459-470, 2001.-   Sun et al., Int. J. Dev. Biol., 42:733-736, 1998.-   Tam et al., J. Am. Chem. Soc., 105:6442, 1983.-   Tkachenko and Simons, J. Biol. Chem., 277:19946-19951, 2002.-   Tonn et al., Anticancer Res., 18:2599-2605, 1998.-   Tumova et al., J. Biol. Chem., 275:9410-9417, 2000.-   Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986.-   Urbinati et al., Atherosc., Thrombosis & Vasc. Biol., 25: 2315-2320,    2005-   van der Flier and Sonnenberg, Cell Tissue Res., 305:285-298, 2001.-   Vita et al., Biopolymers, 47:93-100, 1998.-   Weisshoff et al., Eur. J. Biochem., 259(3):776-788, 1999.-   Wiksten et al., Int. J. Cancer, 95:1-6, 2001.-   Wong et al., Gene, 10:87-94, 1980.-   Woods and Couchman, Curr. Opin. Cell Biol., 13:578-583, 2001.-   Woods and Couchman, Mol. Biol. Cell, 5:183-192, 1994.-   Woods et al., Embo J., 5:665-670, 1986.-   Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987.-   Wu and Wu, Biochemistry, 27:887-892, 1988.-   Xiong et al., Science, 294:339-345, 2001.-   Xue et al., Cancer Res., 57:1682-1689, 1997.-   Yamashita et al., J. Immunol., 162:5940-5948, 1999.-   Yan et al. J. Biol. Chem., 275:7249-7260, 2000.

1. An isolated and purified peptide or polypeptide segment consisting ofbetween 5 and 100 amino acid residues and comprising SEQ ID NO:21 or SEQID NO:13, wherein the segment is not SEQ ID NO:28.
 2. The isolated andpurified peptide or polypeptide of claim 1, wherein said peptide orpolypeptide is 10, 15, 20, 25, 30, 35, 40, 45, 49, 50, 55, 60, 65, 70,75, 80, 85, 90, 95 or 100 amino acid residues in length.
 3. The isolatedand purified peptide or polypeptide of claim 1, wherein said peptide orpolypeptide is between 10 and 80 amino acid residues in length.
 4. Theisolated and purified peptide or polypeptide of claim 1, wherein saidpeptide or polypeptide is between 20 and 50 amino acid residues inlength.
 5. The isolated and purified peptide or polypeptide of claim 1,wherein said peptide or polypeptide is between 30 and 40 amino acidresidues in length.
 6. The isolated and purified peptide of claim 1,wherein said peptide consists of SEQ ID NO:10.
 7. The isolated andpurified peptide of claim 1, wherein said peptide comprises at least 35contiguous amino acids from SEQ ID NO:10.
 8. The isolated and purifiedpeptide of claim 1, wherein said peptide consists of SEQ ID NO:28. 9.The isolated and purified peptide of claim 1, wherein said peptideconsists of SEQ ID NO:21.
 10. A nucleic acid encoding a peptide orpolypeptide segment consisting of between 5 and 100 amino acid residuesand comprising SEQ ID NO:21 or SEQ ID NO:13, wherein the segment is notSEQ ID NO:28. 11-19. (canceled)
 20. The isolated and purified peptide ofclaim 1, dispersed in a pharmaceutically acceptable buffer or diluent.21. A method of inhibiting interaction of α_(v)β₃ or α_(v)β₅ integrinwith syndecan-1 comprising contacting a α_(v)β₃ or α_(v)β₅ integrinmolecule with a peptide or polypeptide segment consisting of between 5and 100 amino acid residues and comprising SEQ ID NO:21 or SEQ ID NO:13,wherein the segment is not SEQ ID NO:28.
 22. The method of claim 21,wherein said peptide or polypeptide is 10, 15, 20, 25, 30, 35, 40, 45,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acid residues inlength.
 23. The method of claim 21, wherein said peptide or polypeptideis between 10 and 80 amino acid residues in length.
 24. The method ofclaim 21, wherein said peptide or polypeptide is between 20 and 50 aminoacid residues in length.
 25. The method of claim 21, wherein saidpeptide or polypeptide is between 30 and 40 amino acid residues inlength.
 26. The method of claim 21, wherein said peptide consists of SEQID NO:10.
 27. The method of claim 21, wherein said peptide comprises atleast 35 contiguous amino acids from SEQ ID NO:10.
 28. The method ofclaim 21, wherein said peptide consists of SEQ ID NO:28.
 29. The methodof claim 21, wherein said peptide consists of SEQ ID NO:21.
 30. Themethod of claim 21, wherein said α_(v)β₃ or α_(v)β₅ integrin is locatedon the surface of a cell.
 31. The method of claim 30, wherein said cellis a cancer cell.
 32. The method of claim 31, wherein said cancer cellis a carcinoma, a myeloma, a melanoma or a glioma.
 33. The method ofclaim 31, further comprising contacting said cancer cell with a secondcancer inhibitory agent.
 34. The method of claim 31, wherein said cancercell is a metastatic cancer cell.
 35. A method of inhibiting α_(v)β₃ orα_(v)β₅ integrin activation by syndecan-1 comprising contacting a cellexpressing an α_(v)β₃ or α_(v)β₅ integrin molecule with a peptide orpolypeptide segment consisting of between 5 and 100 amino acid residuesand comprising SEQ ID NO:21 or SEQ ID NO:13, wherein the segment is notSEQ ID NO:28. 36-44. (canceled)
 45. A method of treating a subject witha cancer, cells of which express α_(v)β₃ or α_(v)β₅ integrin, comprisingcontacting said cells with a peptide or polypeptide segment consistingof between 5 and 100 amino acid residues and comprising SEQ ID NO:21 orSEQ ID NO:13, wherein the segment is not SEQ ID NO:28.
 46. The method ofclaim 45, wherein said peptide or polypeptide is 10, 15, 20, 25, 30, 35,40, 45, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acidresidues in length.
 47. The method of claim 45, wherein said peptide orpolypeptide is between 10 and 80 amino acid residues in length.
 48. Themethod of claim 45, wherein said peptide or polypeptide is between 20and 50 amino acid residues in length.
 49. The method of claim 45,wherein said peptide or polypeptide is between 30 and 40 amino acidresidues in length.
 50. The method of claim 45, wherein said peptideconsists of SEQ ID NO:10.
 51. The method of claim 45, wherein saidpeptide comprises at least 35 contiguous amino acids from SEQ ID NO:10.52. The method of claim 45, wherein said peptide consists of SEQ IDNO:28.
 53. The method of claim 45, wherein said peptide consists of SEQID NO:21.
 54. The method of claim 45, wherein said subject is a human.55. The method of claim 45, wherein said cancer is a carcinoma, amyeloma, a melanoma or a glioma.
 56. The method of claim 45, whereinsaid peptide or polypeptide is administered directly to said cancercells, local to said cancer cells, regional to said cancer cells, orsystemically.
 57. The method of claim 45, further comprisingadministering to said subject a second cancer therapy selected fromchemotherapy, radiotherapy, immunotherapy, hormonal therapy, or genetherapy.
 58. A method of inhibiting angiogenesis comprising contactingan endothelial cell expressing an α_(v)β₃ or α_(v)β₅ integrin moleculewith a peptide or polypeptide segment consisting of between 5 and 100amino acid residues and comprising SEQ ID NO:21 or SEQ ID NO:13. 59-65.(canceled)
 66. The method of claim 58, wherein said peptide consists ofSEQ ID NO:21.
 67. A method of treating a subject having a diseasecharacterized by angiogenesis comprising contacting endothelial cellswhich express α_(v)β₃ or α_(v)β₅ integrin and are responsible for saidangiogenesis, with a peptide or polypeptide segment consisting ofbetween 5 and 100 amino acid residues and comprising SEQ ID NO:21 or SEQID NO:13, wherein the disease is not cancer. 68-75. (canceled)
 76. Themethod of claim 67, wherein said disease is an abnormality of thevasculature (atherosclerosis and hemangiomas), of the eye (diabeticretinopathy and retinopathy of prematurity), of the skin (pyogenicgranulomas, psoriasis, warts, scar keloids, allergic edema, ulcers), ofthe uterus and ovary (dysfunctional uterine bleeding, follicular cysts,endometriosis, pre-eclampsia), of the adipose tissue (obesity), of thebones and joints (rheumatoid arthritis, osteophyte formation), andAIDS-related pathologies resulting from TAT protein of the humanimmunodeficiency virus (HIV) activating the avb3 integrin on endothelialcells. 77-80. (canceled)